U.S. patent application number 14/703495 was filed with the patent office on 2015-08-20 for methods of activating clostridial toxins.
The applicant listed for this patent is Allergan, Inc.. Invention is credited to Dean G. Stathakis, Lance E. Steward, Marc F. Verhagen.
Application Number | 20150232905 14/703495 |
Document ID | / |
Family ID | 39717602 |
Filed Date | 2015-08-20 |
United States Patent
Application |
20150232905 |
Kind Code |
A1 |
Verhagen; Marc F. ; et
al. |
August 20, 2015 |
METHODS OF ACTIVATING CLOSTRIDIAL TOXINS
Abstract
The specification discloses modified Clostridial toxins
comprising an exogenous Clostridial toxin di-chain loop protease
cleavage site located within the di-chain loop region;
polynucleotide molecules encoding such modified Clostridial toxins;
method of producing such modified Clostridial toxins, method of
activating such modified Clostridial toxins and methods of
activating recombinantly-expressed Clostridial toxins.
Inventors: |
Verhagen; Marc F.; (Irvine,
CA) ; Stathakis; Dean G.; (Irvine, CA) ;
Steward; Lance E.; (Irvine, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Allergan, Inc. |
Irvine |
CA |
US |
|
|
Family ID: |
39717602 |
Appl. No.: |
14/703495 |
Filed: |
May 4, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13874105 |
Apr 30, 2013 |
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14703495 |
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12669447 |
Jan 15, 2010 |
8486422 |
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PCT/US08/68504 |
Jun 27, 2008 |
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13874105 |
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60952112 |
Jul 26, 2007 |
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Current U.S.
Class: |
435/68.1 |
Current CPC
Class: |
C12P 21/06 20130101;
C12P 21/02 20130101; C07K 14/33 20130101; C07K 2319/50
20130101 |
International
Class: |
C12P 21/06 20060101
C12P021/06 |
Claims
1. A method of activating a modified Clostridial toxin, the method
comprising the step of incubating a modified Clostridial toxin with
a BoNT/A di-chain loop protease under physiological conditions;
wherein the BoNT/A toxin di-chain loop protease is selected from
the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35,
SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO: 38; wherein the modified
Clostridial toxin comprises an exogenous BoNT/A di-chain loop
protease cleavage site and is selected from the group consisting of
a modified BoNT/B, a modified BoNT/C, a modified BoNT/D, a modified
BoNT/E, a modified BoNT/F, a modified BoNT/G, a modified TeNT, a
modified BaNT, a modified BuNT, and a Clostridial toxin chimeric
variant; and wherein cleavage of the modified Clostridial toxin by
the BoNT/A di-chain loop protease converts the modified Clostridial
toxin from its single-chain polypeptide form into its di-chain
form, thereby activating the modified Clostridial toxin.
2. The method of claim 1, wherein the modified Clostridial toxin is
a modified BoNT/B.
3. The method of claim 1, wherein the modified Clostridial toxin is
a modified BoNT/C.
4. The method of claim 1, wherein the modified Clostridial toxin is
a modified BoNT/E.
5. The method of claim 1, wherein the modified Clostridial toxin
chimeric variant.
6. A method of activating a recombinantly expressed Clostridial
toxin, the method comprising the steps of: (a) expressing in a cell
a polynucleotide molecule encoding a modified Clostridial toxin,
wherein the modified Clostridial toxin comprises an exogenous
BoNT/A di-chain loop protease cleavage site and is selected from
the group consisting of a modified BoNT/B, a modified BoNT/C, a
modified BoNT/D, a modified BoNT/E, a modified BoNT/F, a modified
BoNT/G, a modified TeNT, a modified BaNT, a modified BuNT, and a
Clostridial toxin chimeric variant; (b) purifying the modified
Clostridial toxin; (c) incubating the purified modified Clostridial
toxin with a BoNT/A di-chain loop protease under physiological
conditions; wherein the BoNT/A toxin di-chain loop protease is
selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO: 38; and
wherein cleavage of the modified Clostridial toxin by the BoNT/A
di-chain loop protease converts the modified Clostridial toxin from
its single-chain polypeptide form into its di-chain form, thereby
activating the modified Clostridial toxin.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/874,105, filed Apr. 30, 2013, which is a
continuation of U.S. patent application Ser. No. 12/669,447, filed
Jan. 15, 2010, now U.S. Pat. No. 8,486,422, which claims priority
pursuant to 35 U.S.C. 371 to application PCT/US08/68504, filed Jun.
27, 2008, which claims priority pursuant to 35 U.S.C. .sctn.119(e)
to U.S. Provisional Patent Application Ser. No. 60/952,112 filed
Jul. 26, 2007, all incorporated entirely by reference.
[0002] The ability of Clostridial toxins, such as, e.g., Botulinum
neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,
BoNT/F and BoNT/G, Tetanus neurotoxin (TeNT), Baratium neurotoxin
(BaNT) and Butyricum neurotoxin (BuNT) to inhibit neuronal
transmission are being exploited in a wide variety of therapeutic
and cosmetic applications, see e.g., William J. Lipham, COSMETIC
AND CLINICAL APPLICATIONS OF BOTULINUM TOXIN (Slack, Inc., 2004).
Clostridial toxins commercially available as pharmaceutical
compositions include, BoNT/A preparations, such as, e.g.,
BOTOX.RTM. (Allergan, Inc., Irvine, Calif.),
Dysport.RTM./Reloxin.RTM., (Beaufour Ipsen, Porton Down, England),
Linurase.RTM. (Prollenium, Inc., Ontario, Canada), Neuronox.RTM.
(Medy-Tox, Inc., Ochang-myeon, South Korea) BTX-A (Lanzhou
Institute Biological Products, China) and Xeomin.RTM. (Merz
Pharmaceuticals, GmbH., Frankfurt, Germany); and BoNT/B
preparations, such as, e.g., MyoBloc.TM./NeuroBloc.TM. (Elan
Pharmaceuticals, San Francisco, Calif.). As an example, BOTOX.RTM.
is currently approved in one or more countries for the following
indications: achalasia, adult spasticity, anal fissure, back pain,
blepharospasm, bruxism, cervical dystonia, essential tremor,
glabellar lines or hyperkinetic facial lines, headache, hemifacial
spasm, hyperactivity of bladder, hyperhidrosis, juvenile cerebral
palsy, multiple sclerosis, myoclonic disorders, nasal labial lines,
spasmodic dysphonia, strabismus and VII nerve disorder.
[0003] The increasing use of Clostridial toxin therapies in
treating a wider range of human afflictions necessitates increasing
the efficiency with which these toxins are produced. However,
meeting the needs for the ever increasing demand for such toxin
treatments may become difficult. One outstanding problem is that
all Clostridial toxins need to be converted into the di-chain form
of the molecule in order to achieve optimal activity. Historically,
this conversion has been done in one of two ways. The first method
simply purifies a Clostridial toxin di-chain from the bacterial
strain itself, thereby relying on the naturally-occurring
endogenous protease used to convert the single-chain form of the
toxin into the di-chain form. The second method utilizes an
exogenous protease that converts the single-chain form into the
di-chain by either taking advantage of a fortuitous cleavage site
found in the appropriate location or by genetically engineering a
protease cleavage site of commonly used, commercially available
exogenous proteases. However, there are several drawbacks to both
of these methods. For example, methods employing an endogenous
protease produce low toxin yields because native Clostridial
strains usually produce little toxin. In addition these strains are
poorly suited for research, thus hindering the efforts to genetic
manipulation Clostridial toxins to improve their therapeutic and
cosmetic attributes. Lastly, several Clostridial strains do not
produce the endogenous protease necessary to convert the
single-chain form of the toxin to the di-chain form. A drawback to
the use of exogenous proteases is a lack of protease specificity
that results in inactive toxin because of proteolytic cleavage in
inappropriate locations. In addition, many of the currently
available proteases are from animal sources that lack Good
Manufacture Standard (GMS) approval, requiring additional
purification steps during the manufacturing process. Thus, methods
currently used to convert the single-chain form of the toxin into
the di-chain form are inefficient, cumbersome and/or lead to higher
overall production costs. These drawbacks represent a significant
obstacle to the overall commercial production of Clostridial toxins
and are thus a major problem since di-chain forms of these toxins
are needed for scientific, therapeutic and cosmetic applications.
In addition, both the amount of Clostridial toxins anticipated for
future therapies and the demand for toxins with enhanced
therapeutic properties are increasing. Therefore, there is a need
to develop better methods for producing Clostridial toxin di-chain
molecules in order to meet this need.
[0004] The present invention provides modified Clostridial toxins
that rely on a novel method of converting the single-chain form of
the toxin into the di-chain form and novel methods of convering
single-chain Clostridial toxins. These and related advantages are
useful for various clinical, therapeutic and cosmetic applications,
such as, e.g., the treatment of neuromuscular disorders,
neuropathic disorders, eye disorders, pain, muscle injuries,
headache, cardiovascular diseases, neuropsychiatric disorders,
endocrine disorders, cancers, otic disorders and hyperkinetic
facial lines, as well as, other disorders where a Clostridial toxin
administration to a mammal can produce a beneficial effect.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 shows a schematic of the current paradigm of
Clostridial toxin posttranslational processing. Clostridial toxins
are translated as a single-chain polypeptide of approximately 150
kDa comprising an enzymatic domain, a translocation domain and a
binding domain. A disulfide bridge formed from a cysteine residue
in the enzymatic domain and a cysteine residue from the
translocation domain form a di-chain loop. Within this di-chain
loop is a protease cleavage site for a naturally-occurring protease
that can be produced endogenously from the Clostridial strain
synthesizing the toxin, or exogenously from a source found in the
environment. Cleavage of the protease cleavage site by the
naturally-occurring protease converts the single-chain form of the
toxin into the di-chain form. The di-chain form of the toxin is
held together by the disulfide bond and non-covalent interactions
between the two chains.
[0006] FIGS. 2a and 2b show schematics of the current paradigm of
neurotransmitter release and Clostridial toxin intoxication in a
central and peripheral neuron. FIG. 2a shows a schematic for the
neurotransmitter release mechanism of a central and peripheral
neuron. The release process can be described as comprising two
steps: 1) vesicle docking, where the vesicle-bound SNARE protein of
a vesicle containing neurotransmitter molecules associates with the
membrane-bound SNARE proteins located at the plasma membrane; and
2) neurotransmitter release, where the vesicle fuses with the
plasma membrane and the neurotransmitter molecules are exocytosed.
FIG. 2b shows a schematic of the intoxication mechanism for tetanus
and botulinum toxin activity in a central and peripheral neuron.
This intoxication process can be described as comprising four
steps: 1) receptor binding, where a Clostridial toxin binds to a
Clostridial receptor system and initiates the intoxication process;
2) complex internalization, where after toxin binding, a vesicle
containing the toxin/receptor system complex is endocytosed into
the cell; 3) light chain translocation, where multiple events
result in the release of the active light chain into the cytoplasm;
and 4) enzymatic target modification, where the active light chain
of Clostridial toxin proteolytically cleaves its target SNARE
substrate, such as, e.g., SNAP-25, VAMP or Syntaxin, thereby
preventing vesicle docking and neurotransmitter release.
DETAILED DESCRIPTION
[0007] Clostridia toxins produced by Clostridium botulinum,
Clostridium tetani, Clostridium baratii and Clostridium butyricum
are the most widely used in therapeutic and cosmetic treatments of
humans and other mammals. Strains of C. botulinum produce seven
antigenically-distinct types of Botulinum toxins (BoNTs), which
have been identified by investigating botulism outbreaks in man
(BoNT/A, /B, /E and /F), animals (BoNT/C1 and /D), or isolated from
soil (BoNT/G). BoNTs possess approximately 35% amino acid identity
with each other and share the same functional domain organization
and overall structural architecture. It is recognized by those of
skill in the art that within each type of Clostridial toxin there
can be subtypes that differ somewhat in their amino acid sequence,
and also in the nucleic acids encoding these proteins. For example,
there are presently four BoNT/A subtypes, BoNT/A1, BoNT/A2, BoNT/A3
and BoNT/A4, with specific subtypes showing approximately 89% amino
acid identity when compared to another BoNT/A subtype. While all
seven BoNT serotypes have similar structure and pharmacological
properties, each also displays heterogeneous bacteriological
characteristics. In contrast, tetanus toxin (TeNT) is produced by a
uniform group of C. tetani. Two other species of Clostridia, C.
baratii and C. butyricum, also produce toxins, BaNT and BuNT
respectively, which are similar to BoNT/F and BoNT/E,
respectively.
[0008] Clostridial toxins are each translated as a single chain
polypeptide of approximately 150 kDa that is subsequently cleaved
by proteolytic scission within a disulfide loop by a
naturally-occurring protease (FIG. 1). This cleavage occurs within
the discrete di-chain loop region created between two cysteine
residues that form a disulfide bridge. This posttranslational
processing yields a di-chain molecule comprising an approximately
50 kDa light chain (LC) and an approximately 100 kDa heavy chain
(HC) held together by the single disulfide bond and non-covalent
interactions between the two chains. The naturally-occurring
protease used to convert the single chain molecule into the
di-chain is currently not known. In some bacterial serotypes, such
as, e.g., a BoNT/A, a BoNT/B proteolytic, a BoNT/F proteolytic, a
BaNT proteolytic strain, or a TeNT, the naturally-occurring
protease is produced endogenously by the bacteria serotype and
cleavage occurs within the cell before the toxin is release into
the environment. However, in other bacterial serotypes, such as,
e.g., a BoNT/B nonproteolytic, a BoNT/C1, a BoNT/D, a BoNT/E, a
BoNT/F nonproteolytic, a BoNT/G, a BaNT nonproteolytic, or a BuNT,
the bacterial strain appears not to produce appreciable amounts of
an endogenous protease capable of converting the single chain form
of the toxin into the di-chain form. In these situations, the toxin
is released from the cell as a single-chain toxin which is
subsequently converted into the di-chain form by a
naturally-occurring protease found in the environment.
[0009] The present invention discloses novel methods that can
convert the single-chain polypeptide form of a
recombinantly-expressed Clostridial toxin or a modified Clostridial
toxins into the di-chain form using the enzymatic activity of a
di-chain loop protease isolated from a Clostridial bacteria strain.
The present specification discloses several proteases having
proteolytic activity for the cleavage site within the di-chain loop
region. Thus discovery has lead to the development of methods of
activating recombinantly-expressed Clostridial toxins, irrespective
of whether these recombinantly-expressed Clostridial toxins are 1)
Clostridial toxins capable of cleavage by a di-chain protease
expressed within the Clostridial bacterial strain expressing that
toxin, such as, e.g., a BoNT/A, a BoNT/B proteolytic, a BoNT/F
proteolytic, a BaNT proteolytic, or a TeNT baterical strain; or 2)
modified Clostridial toxins comprising a cleavage site within the
di-chain loop region that can be cleaved the di-chain proteases
disclosed, such as, e.g., a Clostridial toxin from a BoNT/B
nonproteolytic, a BoNT/C1, a BoNT/D, a BoNT/E, a BoNT/F
nonproteolytic, a BoNT/G, a BaNT nonproteolytic, or BuNT, bacterial
strain modified to include the di-chain loop region from a
Clostridial toxin produced by a BoNT/A, a BoNT/B proteolytic or a
BoNT/F proteolytic bacterial strain.
[0010] As a non-limiting example, BoNT/A expressed naturally from
Clostridia botulinum serotype A strain is produced in its di-chain
polypeptide form. This is because the single-chain polypeptide is
converted into its di-chain form by a di-chain loop protease
produced by the bacterium. However, when a BoNT/A is recombinantly
expresses, such as, e.g., in an E. coli bacterial strain, this
conversion does not occur since E. coli strains do not express the
di-chain loop protease. As a result, recombinantly expresses BoNT/A
is primarily isolated in its single-chain polypeptide form, a form
that is approximately 100 times less active than the di-chain form.
Thus, the presently disclosed methods of activating
recombinantly-expressed Clostridial toxins include methods that
convert a recombinantly expressed single-chain BoNT/A into its
di-chain form using a BoNT/A di-chain protease disclosed in the
present specification.
[0011] As another non-limiting example, BoNT/E expressed naturally
from Clostridia botulinum serotype E strain is produced in its
single-chain polypeptide form. This is because the C. botulinum
serotype E bacterium does not express a di-chain loop protease
capable of cleaving the toxin within the di-chain loop region.
Similarly, when a BoNT/E is recombinantly expresses, such as, e.g.,
in an E. coli bacterial strain, this conversion does not occur
since E. coli strains also do not express the di-chain loop
protease. Thus, the present specification discloses modified
Clostridial toxins comprising a di-chain loop protease cleavage
site from a Clostridial toxin expressed in a Clostridia botulinum
strain expressing an endogenous di-chain loop protease, such as,
e.g., a modified BoNT/E comprising a BoNT/A di-chain loop region
including a BoNT/A di-chain loop protease cleavage site. This can
be accomplished, for instance, by replacing the naturally-occurring
di-chain loop region from BoNT/E with a di-chain loop region
including the di-chain loop protease cleavage site from BoNT/A.
Using such a modified BoNT/E, the presently disclosed methods of
activating recombinantly-expressed Clostridial toxins include
methods that convert a recombinantly expressed single-chain
modified BoNT/E into its di-chain form using a BoNT/A di-chain
protease disclosed in the present specification.
[0012] Aspects of the present invention provide modified
Clostridial toxins comprising an exogenous Clostridial toxin
di-chain loop including a Clostridial toxin di-chain loop protease
cleavage site from a different Clostridial toxin. It is envisioned
that the exogenous di-chain loop region can replace the endogenous
di-chain loop region or be in addition to the endogenous di-chain
loop region. It is also envisioned that any Clostridial toxin
di-chain loop region including a di-chain loop protease cleavage
site can be used. including, without limitation, a BoNT/A di-chain
loop region including a di-chain loop protease cleavage site, a
BoNT/B di-chain loop region including a di-chain loop protease
cleavage site, a BoNT/C1 di-chain loop region including a di-chain
loop protease cleavage site, a BoNT/D di-chain loop region
including a di-chain loop protease cleavage site, a BoNT/E di-chain
loop region including a di-chain loop protease cleavage site, a
BoNT/F di-chain loop region including a di-chain loop protease
cleavage site, a BoNT/G di-chain loop region including a di-chain
loop protease cleavage site, a TeNT di-chain loop region including
a di-chain loop protease cleavage site, a BaNT di-chain loop region
including a di-chain loop protease cleavage site and a BuNT
di-chain loop region including a di-chain loop protease cleavage
site.
[0013] Other aspects of the present invention provide
polynucleotide molecules encoding modified Clostridial toxins
comprising an exogenous Clostridial toxin di-chain loop including a
Clostridial toxin di-chain loop protease cleavage site from a
different Clostridial toxin.
[0014] Other aspects of the present invention provide methods of
producing a modified Clostridial toxin comprising an exogenous
Clostridial toxin di-chain loop including a Clostridial toxin
di-chain loop protease cleavage site from a different Clostridial
toxin. Other aspects of the present invention provide methods of
producing in a cell a modified Clostridial toxin comprising an
exogenous Clostridial toxin di-chain loop including a Clostridial
toxin di-chain loop protease cleavage site from a different
Clostridial toxin.
[0015] Other aspects of the present invention provide methods of
activating a modified Clostridial toxin comprising an exogenous
Clostridial toxin di-chain loop including a Clostridial toxin
di-chain loop protease cleavage site from a different Clostridial
toxin.
[0016] Yet other aspects of the present invention provide methods
of activating a modified Clostridial toxin comprising an exogenous
Clostridial toxin di-chain loop including a Clostridial toxin
di-chain loop protease cleavage site from a different Clostridial
toxin.
[0017] Each mature di-chain molecule comprises three functionally
distinct domains: 1) an enzymatic domain located in the LC that
includes a metalloprotease region containing a zinc-dependent
endopeptidase activity which specifically targets core components
of the neurotransmitter release apparatus; 2) a translocation
domain contained within the amino-terminal half of the HC (H.sub.N)
that facilitates release of the LC from intracellular vesicles into
the cytoplasm of the target cell; and 3) a binding domain found
within the carboxyl-terminal half of the HC (H.sub.C) that
determines the binding activity and binding specificity of the
toxin to the receptor complex located at the surface of the target
cell. The H.sub.C domain comprises two distinct structural features
of roughly equal size that indicate function and are designated the
H.sub.CN and H.sub.CC subdomains. Table 1 gives approximate
boundary regions for each domain found in exemplary Clostridial
toxins.
TABLE-US-00001 TABLE 1 Clostridial Toxin Reference Sequences and
Regions H.sub.C Toxin SEQ ID NO: LC H.sub.N H.sub.CN H.sub.CC
BoNT/A 1 M1-K448 A449-I873 I874-P1110 Y1111-L1296 BoNT/B 2 M1-K441
A442-I860 L861-E1097 Y1098-E1291 BoNT/C1 3 M1-K449 T450-I868
N869-E1111 Y1112-E1291 BoNT/D 4 M1-R445 D446-I864 N865-E1098
Y1099-E1276 BoNT/E 5 M1-R422 K423-I847 K848-E1085 Y1086-K1252
BoNT/F 6 M1-K439 A440-I866 K867-K1105 Y1106-E1274 BoNT/G 7 M1-K446
S447-I865 S866-Q1105 Y1106-E1297 TeNT 8 M1-A457 S458-L881
K882-E1127 Y1128-D1315 BaNT 9 M1-K431 N432-I857 I858-K1094
Y1095-E1268 BuNT 10 M1-R422 K423-I847 K848-E1085 Y1086-K1251
[0018] The binding, translocation and enzymatic activity of these
three functional domains are all necessary for toxicity. While all
details of this process are not yet precisely known, the overall
cellular intoxication mechanism whereby Clostridial toxins enter a
neuron and inhibit neurotransmitter release is similar, regardless
of type. Although the applicants have no wish to be limited by the
following description, the intoxication mechanism can be described
as comprising at least four steps: 1) receptor binding, 2) complex
internalization, 3) light chain translocation, and 4) enzymatic
target modification (see FIG. 2). The process is initiated when the
H.sub.C domain of a Clostridial toxin binds to a toxin-specific
receptor complex located on the plasma membrane surface of a target
cell. The binding specificity of a receptor complex is thought to
be achieved, in part, by specific combinations of gangliosides and
protein receptors that appear to distinctly comprise each
Clostridial toxin receptor complex. Once bound, the toxin/receptor
complexes are internalized by endocytosis and the internalized
vesicles are sorted to specific intracellular routes. The
translocation step appears to be triggered by the acidification of
the vesicle compartment. This process seems to initiate two
important pH-dependent structural rearrangements that increase
hydrophobicity and promote formation di-chain form of the toxin.
Once activated, light chain endopeptidase of the toxin is released
from the intracellular vesicle into the cytosol where it
specifically targets one of three known core components of the
neurotransmitter release apparatus. These core proteins,
vesicle-associated membrane protein (VAMP)/synaptobrevin,
synaptosomal-associated protein of 25 kDa (SNAP-25) and Syntaxin,
are necessary for synaptic vesicle docking and fusion at the nerve
terminal and constitute members of the soluble
N-ethylmaleimide-sensitive factor-attachment protein-receptor
(SNARE) family. BoNT/A and BoNT/E cleave SNAP-25 in the
carboxyl-terminal region, releasing a nine or twenty-six amino acid
segment, respectively, and BoNT/C1 also cleaves SNAP-25 near the
carboxyl-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F
and BoNT/G, and tetanus toxin, act on the conserved central portion
of VAMP, and release the amino-terminal portion of VAMP into the
cytosol. BoNT/C1 cleaves syntaxin at a single site near the
cytosolic membrane surface. The selective proteolysis of synaptic
SNAREs accounts for the block of neurotransmitter release caused by
Clostridial toxins in vivo. The SNARE protein targets of
Clostridial toxins are common to exocytosis in a variety of
non-neuronal types; in these cells, as in neurons, light chain
peptidase activity inhibits exocytosis, see, e.g., Yann Humeau et
al., How Botulinum and Tetanus Neurotoxins Block Neurotransmitter
Release, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al.,
Botulinum and Tetanus Neurotoxins: Structure, Function and
Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002);
Giovanna Lalli et al., The Journey of Tetanus and Botulinum
Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437,
(2003).
[0019] Aspects of the present invention provide, in part, a
Clostridial toxin. As used herein, the term "Clostridial toxin"
means any polypeptide that can execute the overall cellular
mechanism whereby a Clostridial toxin enters a neuron and inhibits
neurotransmitter release and encompasses the binding of a
Clostridial toxin to a low or high affinity receptor complex, the
internalization of the toxin/receptor complex, the translocation of
the Clostridial toxin light chain into the cytoplasm and the
enzymatic modification of a Clostridial toxin substrate.
[0020] A Clostridial toxin includes, without limitation, naturally
occurring Clostridial toxin variants, such as, e.g., Clostridial
toxin isoforms and Clostridial toxin subtypes; non-naturally
occurring Clostridial toxin variants, such as, e.g., conservative
Clostridial toxin variants, non-conservative Clostridial toxin
variants, Clostridial toxin chimeric variants and active
Clostridial toxin fragments thereof, or any combination thereof. As
used herein, the term "Clostridial toxin variant," whether
naturally-occurring or non-naturally-occurring, means a Clostridial
toxin that has at least one amino acid change from the
corresponding region of the disclosed reference sequences (see
Table 1) and can be described in percent identity to the
corresponding region of that reference sequence. As non-limiting
examples, a BoNT/A variant comprising amino acids 1-1296 of SEQ ID
NO: 1 will have at least one amino acid difference, such as, e.g.,
an amino acid substitution, deletion or addition, as compared to
the amino acid region 1-1296 of SEQ ID NO: 1; a BoNT/B variant
comprising amino acids 1-1291 of SEQ ID NO: 2 will have at least
one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid
region 1-1291 of SEQ ID NO: 2; a BoNT/C1 variant comprising amino
acids 1-1291 of SEQ ID NO: 3 will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the amino acid region 1-1291 of SEQ ID NO:
3; a BoNT/D variant comprising amino acids 1-1276 of SEQ ID NO: 4
will have at least one amino acid difference, such as, e.g., an
amino acid substitution, deletion or addition, as compared to the
amino acid region 1-1276 of SEQ ID NO: 4; a BoNT/E variant
comprising amino acids 1-1252 of SEQ ID NO: 5 will have at least
one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid
region 1-1252 of SEQ ID NO: 5; a BoNT/F variant comprising amino
acids 1-1274 of SEQ ID NO: 6 will have at least one amino acid
difference, such as, e.g., an amino acid substitution, deletion or
addition, as compared to the amino acid region 1-1274 of SEQ ID NO:
6; a BoNT/G variant comprising amino acids 1-1297 of SEQ ID NO: 7
will have at least one amino acid difference, such as, e.g., an
amino acid substitution, deletion or addition, as compared to the
amino acid region 1-1297 of SEQ ID NO: 7; a TeNT variant comprising
amino acids 1-1315 of SEQ ID NO: 8 will have at least one amino
acid difference, such as, e.g., an amino acid substitution,
deletion or addition, as compared to the amino acid region 1-1315
of SEQ ID NO: 8; a BaNT variant comprising amino acids 1-1268 of
SEQ ID NO: 9 will have at least one amino acid difference, such as,
e.g., an amino acid substitution, deletion or addition, as compared
to the amino acid region 1-1268 of SEQ ID NO: 9; and a BuNT variant
comprising amino acids 1-1251 of SEQ ID NO: 10 will have at least
one amino acid difference, such as, e.g., an amino acid
substitution, deletion or addition, as compared to the amino acid
region 1-1251 of SEQ ID NO: 10.
[0021] Any of a variety of sequence alignment methods can be used
to determine percent identity, including, without limitation,
global methods, local methods and hybrid methods, such as, e.g.,
segment approach methods. Protocols to determine percent identity
are routine procedures within the scope of one skilled in the art
and from the teaching herein.
[0022] Global methods align sequences from the beginning to the end
of the molecule and determine the best alignment by adding up
scores of individual residue pairs and by imposing gap penalties.
Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D.
Thompson et al., CLUSTAL W: Improving the Sensitivity of
Progressive Multiple Sequence Alignment Through Sequence Weighting,
Position-Specific Gap Penalties and Weight Matrix Choice, 22(22)
Nucleic Acids Research 4673-4680 (1994); and iterative refinement,
see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of
Multiple Protein Sequence Alignments by Iterative Refinement as
Assessed by Reference to Structural Alignments, 264(4) J. Mol.
Biol. 823-838 (1996).
[0023] Local methods align sequences by identifying one or more
conserved motifs shared by all of the input sequences. Non-limiting
methods include, e.g., Match-box, see, e.g., Eric Depiereux and
Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the
Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS
501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al.,
Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for
Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see,
e.g., Ivo Van Walle et al., Align-M--A New Algorithm for Multiple
Alignment of Highly Divergent Sequences, 20(9)
Bioinformatics:1428-1435 (2004).
[0024] Hybrid methods combine functional aspects of both global and
local alignment methods. Non-limiting methods include, e.g.,
segment-to-segment comparison, see, e.g., Burkhard Morgenstern et
al., Multiple DNA and Protein Sequence Alignment Based On
Segment-To-Segment Comparison, 93(22) Proc. Natl. Acad. Sci. U.S.A.
12098-12103 (1996); T-Coffee, see, e.g., Cedric Notredame et al.,
T-Coffee: A Novel Algorithm for Multiple Sequence Alignment, 302(1)
J. Mol. Biol. 205-217 (2000); MUSCLE, see, e.g., Robert C. Edgar,
MUSCLE: Multiple Sequence Alignment With High Score Accuracy and
High Throughput, 32(5) Nucleic Acids Res. 1792-1797 (2004); and
DIALIGN-T, see, e.g., Amarendran R Subramanian et al., DIALIGN-T:
An Improved Algorithm for Segment-Based Multiple Sequence
Alignment, 6(1) BMC Bioinformatics 66 (2005).
[0025] As used herein, the term "naturally occurring Clostridial
toxin variant" means any Clostridial toxin produced without the aid
of any human manipulation, including, without limitation,
Clostridial toxin isoforms produced from alternatively-spliced
transcripts, Clostridial toxin isoforms produced by spontaneous
mutation and Clostridial toxin subtypes. Non-limiting examples of a
Clostridial toxin isoform include, e.g., BoNT/A isoforms, BoNT/B
isoforms, BoNT/C1 isoforms, BoNT/D isoforms, BoNT/E isoforms,
BoNT/F isoforms, BoNT/G isoforms, TeNT isoforms, BaNT isoforms, and
BuNT isoforms. Non-limiting examples of a Clostridial toxin subtype
include, e.g., BoNT/A subtypes BoNT/A1, BoNT/A2, BoNT/A3 and
BoNT/A4; BoNT/B subtypes BoNT/B1, BoNT/B2, BoNT/B bivalent and
BoNT/B nonproteolytic; BoNT/C1 subtypes BoNT/C1-1 and BoNT/C1-2;
BoNT/E subtypes BoNT/E1, BoNT/E2 and BoNT/E3; and BoNT/F subtypes
BoNT/F1, BoNT/F2, BoNT/F3 and BoNT/F4.
[0026] As used herein, the term "non-naturally occurring
Clostridial toxin variant" means any Clostridial toxin produced
with the aid of human manipulation, including, without limitation,
Clostridial toxins produced by genetic engineering using random
mutagenesis or rational design and Clostridial toxins produced by
chemical synthesis. Non-limiting examples of non-naturally
occurring Clostridial toxin variants include, e.g., conservative
Clostridial toxin variants, non-conservative Clostridial toxin
variants, Clostridial toxin chimeric variants and active
Clostridial toxin fragments.
[0027] As used herein, the term "conservative Clostridial toxin
variant" means a Clostridial toxin that has at least one amino acid
substituted by another amino acid or an amino acid analog that has
at least one property similar to that of the original amino acid
from the reference Clostridial toxin sequence (Table 1). Examples
of properties include, without limitation, similar size,
topography, charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity, hydrogen-bonding capacity, a
physicochemical property, of the like, or any combination thereof.
A conservative Clostridial toxin variant can function in
substantially the same manner as the reference Clostridial toxin on
which the conservative Clostridial toxin variant is based, and can
be substituted for the reference Clostridial toxin in any aspect of
the present invention. A conservative Clostridial toxin variant may
substitute one or more amino acids, two or more amino acids, three
or more amino acids, four or more amino acids, five or more amino
acids, ten or more amino acids, 20 or more amino acids, 30 or more
amino acids, 40 or more amino acids, 50 or more amino acids, 100 or
more amino acids, 200 or more amino acids, 300 or more amino acids,
400 or more amino acids, or 500 or more amino acids from the
reference Clostridial toxin on which the conservative Clostridial
toxin variant is based. A conservative Clostridial toxin variant
can also substitute at least 10 contiguous amino acids, at least 15
contiguous amino acids, at least 20 contiguous amino acids, or at
least 25 contiguous amino acids from the reference Clostridial
toxin on which the conservative Clostridial toxin variant is based,
that possess at least 50% amino acid identity, 65% amino acid
identity, 75% amino acid identity, 85% amino acid identity or 95%
amino acid identity to the reference Clostridial toxin on which the
conservative Clostridial toxin variant is based. Non-limiting
examples of a conservative Clostridial toxin variant include, e.g.,
conservative BoNT/A variants, conservative BoNT/B variants,
conservative BoNT/C1 variants, conservative BoNT/D variants,
conservative BoNT/E variants, conservative BoNT/F variants,
conservative BoNT/G variants, conservative TeNT variants,
conservative BaNT variants and conservative BuNT variants.
[0028] As used herein, the term "non-conservative Clostridial toxin
variant" means a Clostridial toxin in which 1) at least one amino
acid is deleted from the reference Clostridial toxin on which the
non-conservative Clostridial toxin variant is based; 2) at least
one amino acid added to the reference Clostridial toxin on which
the non-conservative Clostridial toxin is based; or 3) at least one
amino acid is substituted by another amino acid or an amino acid
analog that does not share any property similar to that of the
original amino acid from the reference Clostridial toxin sequence
(Table 1). A non-conservative Clostridial toxin variant can
function in substantially the same manner as the reference
Clostridial toxin on which the non-conservative Clostridial toxin
variant is based, and can be substituted for the reference
Clostridial toxin in any aspect of the present invention. A
non-conservative Clostridial toxin variant can delete one or more
amino acids, two or more amino acids, three or more amino acids,
four or more amino acids, five or more amino acids, and ten or more
amino acids from the reference Clostridial toxin on which the
non-conservative Clostridial toxin variant is based. A
non-conservative Clostridial toxin variant can add one or more
amino acids, two or more amino acids, three or more amino acids,
four or more amino acids, five or more amino acids, and ten or more
amino acids to the reference Clostridial toxin on which the
non-conservative Clostridial toxin variant is based. A
non-conservative Clostridial toxin variant may substitute one or
more amino acids, two or more amino acids, three or more amino
acids, four or more amino acids, five or more amino acids, ten or
more amino acids, 20 or more amino acids, 30 or more amino acids,
40 or more amino acids, 50 or more amino acids, 100 or more amino
acids, 200 or more amino acids, 300 or more amino acids, 400 or
more amino acids, or 500 or more amino acids from the reference
Clostridial toxin on which the non-conservative Clostridial toxin
variant is based. A non-conservative Clostridial toxin variant can
also substitute at least 10 contiguous amino acids, at least 15
contiguous amino acids, at least 20 contiguous amino acids, or at
least 25 contiguous amino acids from the reference Clostridial
toxin on which the non-conservative Clostridial toxin variant is
based, that possess at least 50% amino acid identity, 65% amino
acid identity, 75% amino acid identity, 85% amino acid identity or
95% amino acid identity to the reference Clostridial toxin on which
the non-conservative Clostridial toxin variant is based.
Non-limiting examples of a non-conservative Clostridial toxin
variant include, e.g., non-conservative BoNT/A variants,
non-conservative BoNT/B variants, non-conservative BoNT/C1
variants, non-conservative BoNT/D variants, non-conservative BoNT/E
variants, non-conservative BoNT/F variants, non-conservative BoNT/G
variants, non-conservative TeNT variants, non-conservative BaNT
variants and non-conservative BuNT variants.
[0029] As used herein, the term "Clostridial toxin chimeric
variant" means a molecule comprising at least a portion of a
Clostridial toxin and at least a portion of at least one other
protein to form a toxin with at least one property different from
the reference Clostridial toxins of Table 1. One class of
Clostridial toxin chimeric variant comprises a modified Clostridial
toxin were the endogenous cell binding domain of a
naturally-occurring Clostridial toxin is either modified or
replaced with a cell binding domain of another molecule. Such
modified Clostridial toxin possesses an altered cell binding
activity because the modified toxin can, e.g., use the same
receptor present on the surface of a naturally occurring
Clostridial toxin target cell, referred to as an enhanced cell
binding activity for a naturally-occurring Clostridial toxin target
cell; use a different receptor present on the surface of a
naturally occurring Clostridial toxin target cell, referred to as
an altered cell binding activity for a naturally-occurring
Clostridial toxin target cell, or use a different receptor present
on the surface of the non-Clostridial toxin target cell, referred
to as an altered cell binding activity for a
non-naturally-occurring Clostridial toxin target cell.
[0030] A Clostridial toxin chimeric variant can be a modified
Clostridial toxin with an enhanced cell binding activity capable of
intoxicating a naturally occurring Clostridial toxin target cell,
e.g., a motor neuron. One way this enhanced binding activity is
achieved by modifying the endogenous targeting domain of a
naturally-occurring Clostridial toxin in order to enhance a cell
binding activity of the toxin for its naturally-occurring receptor.
Such modifications to a targeting domain result in, e.g., a
enhanced cell binding activity that increases binding affinity for
an endogenous Clostridial toxin receptor present on a
naturally-occurring Clostridial toxin target cell; an enhanced cell
binding activity that increases binding specificity for a subgroup
of endogenous Clostridial toxin receptors present on a
naturally-occurring Clostridial toxin target cell; or an enhanced
cell binding activity that increases both binding affinity and
binding specificity. Non-limiting examples of modified Clostridial
toxins an enhanced cell binding activity for a naturally-occurring
Clostridial toxin receptor are described in, e.g., Lance E.
Steward, et al., Modified Clostridial Toxins with Enhanced
Targeting Capabilities For Endogenous Clostridial Toxin Receptors,
International Patent Publication No. 2006/008956 (Mar. 14, 2006),
Lance E. Steward, Modified Clostridial Toxins with Enhanced
Translocation Capability and Enhanced Targeting Activity, U.S.
patent application Ser. No. 11/776,043 (Jul. 11, 2007), each of
which is hereby incorporated by reference in its entirety.
[0031] A Clostridial toxin chimeric variant can be a modified
Clostridial toxin with an altered cell binding activity capable of
intoxicating a naturally occurring Clostridial toxin target cell,
e.g., a motor neuron. One way this altered capability is achieved
by replacing the endogenous targeting domain of a
naturally-occurring Clostridial toxin with a targeting domain of
another molecule that selectively binds to a different receptor
present on the surface of a naturally occurring Clostridial toxin
target cell. Such a modification to a targeting domain results in a
modified toxin that is able to selectively bind to a
non-Clostridial toxin receptor (target receptor) present on a
Clostridial toxin target cell. This enhanced binding activity for a
naturally occurring Clostridial toxin target cell allows for lower
effective doses of a modified Clostridial toxin to be administered
to an individual because more toxin will be delivered to the target
cell. Thus, modified Clostridial toxins with an enhanced binding
activity will reduce the undesirable dispersal of the toxin to
areas not targeted for treatment, thereby reducing or preventing
the undesirable side-effects associated with diffusion of a
Clostridial toxin to an unwanted location. Non-limiting examples of
modified Clostridial toxins with an altered cell binding capability
for a Clostridial toxin target cell are described in, e.g., Lance
E. Steward et al., Modified Clostridial Toxins with Altered
Targeting Capabilities For Clostridial Toxin Target Cells,
International Patent Publication No. 2006/009831 (Mar. 14, 2005);
Lance E. Steward et al., Multivalent Clostridial Toxin Derivatives
and Methods of Their Use, U.S. Patent Publication No. 2006/0211619
(Sep. 21, 2006); and Lance E. Steward, Modified Clostridial Toxins
with Enhanced Translocation Capabilities and Altered Targeting
Activity for Clostridial Toxin Target Cells, U.S. patent
application Ser. No. 11/776,052, (Jul. 11, 2007), each of which is
hereby incorporated by reference in its entirety.
[0032] A Clostridial toxin chimeric variant can be a modified
Clostridial toxin with an altered cell binding activity capable of
intoxicating a cell other than a naturally occurring Clostridial
toxin target cell, e.g., a cell other than a motor neuron. These
modified toxins achieve this intoxication by using a target
receptor present on non-Clostridial toxin target cell. This
re-targeted capability is achieved by replacing a
naturally-occurring targeting domain of a Clostridial toxin with a
targeting domain showing a selective binding activity for a
non-Clostridial toxin receptor present in a non-Clostridial toxin
target cell. Such modifications to a targeting domain result in a
modified toxin that is able to selectively bind to a
non-Clostridial toxin receptor (target receptor) present on a
non-Clostridial toxin target cell (re-targeted). A modified
Clostridial toxin with an altered targeting activity for a
non-Clostridial toxin target cell can bind to a target receptor,
translocate into the cytoplasm, and exert its proteolytic effect on
the SNARE complex of the non-Clostridial toxin target cell.
Non-limiting examples of modified Clostridial toxins with an
altered targeting activity for a non-Clostridial toxin target cell
are described in, e.g., Keith A. Foster et al., Clostridial Toxin
Derivatives Able To Modify Peripheral Sensory Afferent Functions,
U.S. Pat. No. 5,989,545 (Nov. 23, 1999); Clifford C. Shone et al.,
Recombinant Toxin Fragments, U.S. Pat. No. 6,461,617 (Oct. 8,
2002); Conrad P. Quinn et al., Methods and Compounds for the
Treatment of Mucus Hypersecretion, U.S. Pat. No. 6,632,440 (Oct.
14, 2003); Lance E. Steward et al., Methods And Compositions For
The Treatment Of Pancreatitis, U.S. Pat. No. 6,843,998 (Jan. 18,
2005); Stephan Donovan, Clostridial Toxin Derivatives and Methods
For Treating Pain, U.S. Pat. No. 7,138,127 (Nov. 21, 2006); Keith
A. Foster et al., Inhibition of Secretion from Non-Neural Cells,
U.S. Patent Publication 2003/0180289 (Sep. 25, 2003); J. Oliver
Dolly et al., Activatable Recombinant Neurotoxins, U.S. Pat. No.
7,132,259 (Nov. 7, 2006); Keith A. Foster et al., Re-targeted Toxin
Conjugates, International Patent Publication WO 2005/023309 (Mar.
17, 2005); Lance E. Steward et al., Multivalent Clostridial Toxin
Derivatives and Methods of Their Use, U.S. patent application Ser.
No. 11/376,696 (Mar. 15, 2006); Keith A. Foster, Fusion Proteins,
International Patent Publication WO 2006/059093 (Jun. 8, 2005);
Keith A. Foster, Non-Cytotoxic Protein Conjugates, International
Patent Publication WO 2006/059105 (Jun. 8, 2005); and Lance E.
Steward, Modified Clostridial Toxins with Enhanced Translocation
Capabilities and Altered Targeting Capabilities for Non-Clostridial
Toxin Target Cells, U.S. patent application Ser. No. 11/776,075
(Jul. 11, 2007), each of which is hereby incorporated by reference
in its entirety. The ability to re-target the therapeutic effects
associated with Clostridial toxins has greatly extended the number
of medicinal applications able to use a Clostridial toxin therapy.
As a non-limiting example, modified Clostridial toxins retargeted
to sensory neurons are useful in treating various kinds of chronic
pain, such as, e.g., hyperalgesia and allodynia, neuropathic pain
and inflammatory pain, see, e.g., Foster, supra, (1999); and
Donovan, supra, (2006); and Stephan Donovan, Method For Treating
Neurogenic Inflammation Pain with Botulinum Toxin and Substance P
Components, U.S. Pat. No. 7,022,329 (Apr. 4, 2006). As another
non-limiting example, modified Clostridial toxins retargeted to
pancreatic cells are useful in treating pancreatitis, see, e.g.,
Steward, supra, (2005).
[0033] Thus, in an embodiment, a Clostridial toxin chimeric variant
can comprise a modified Clostridial toxin disclosed in the present
specification where the binding domain comprises an enhanced cell
binding activity capable of intoxicating a naturally occurring
Clostridial toxin target cell. In another embodiment, a Clostridial
toxin chimeric variant can comprise a modified Clostridial toxin
disclosed in the present specification where the binding domain
comprises an altered cell binding activity capable of intoxicating
a naturally occurring Clostridial toxin target cell. In still
another embodiment, a Clostridial toxin chimeric variant can
comprise a modified Clostridial toxin disclosed in the present
specification where the binding domain comprises an altered cell
binding activity capable of intoxicating a non-naturally occurring
Clostridial toxin target cell.
[0034] It is also envisioned that any of a variety of Clostridial
toxin fragments can be useful in aspects of the present invention
with the proviso that these active fragments can execute the
overall cellular mechanism whereby a Clostridial toxin
proteolytically cleaves a substrate. Thus, aspects of this
embodiment can include Clostridial toxin fragments having a length
of, e.g., at least 300 amino acids, at least 400 amino acids, at
least 500 amino acids, at least 600 amino acids, at least 700 amino
acids, at least 800 amino acids, at least 900 amino acids, at least
1000 amino acids, at least 1100 amino acids and at least 1200 amino
acids. Other aspects of this embodiment, can include Clostridial
toxin fragments having a length of, e.g., at most 300 amino acids,
at most 400 amino acids, at most 500 amino acids, at most 600 amino
acids, at most 700 amino acids, at most 800 amino acids, at most
900 amino acids, at most 1000 amino acids, at most 1100 amino acids
and at most 1200 amino acids.
[0035] It is also envisioned that any of a variety of Clostridial
toxin fragments comprising the light chain can be useful in aspects
of the present invention with the proviso that these light chain
fragments can specifically target the core components of the
neurotransmitter release apparatus and thus participate in
executing the overall cellular mechanism whereby a Clostridial
toxin proteolytically cleaves a substrate. The light chains of
Clostridial toxins are approximately 420-460 amino acids in length
and comprise an enzymatic domain (Table 1). Research has shown that
the entire length of a Clostridial toxin light chain is not
necessary for the enzymatic activity of the enzymatic domain. As a
non-limiting example, the first eight amino acids of the BoNT/A
light chain (residues 1-8 of SEQ ID NO: 1) are not required for
enzymatic activity. As another non-limiting example, the first
eight amino acids of the TeNT light chain (residues 1-8 of SEQ ID
NO: 8) are not required for enzymatic activity. Likewise, the
carboxyl-terminus of the light chain is not necessary for activity.
As a non-limiting example, the last 32 amino acids of the BoNT/A
light chain (residues 417-448 of SEQ ID NO: 1) are not required for
enzymatic activity. As another non-limiting example, the last 31
amino acids of the TeNT light chain (residues 427-457 of SEQ ID NO:
8) are not required for enzymatic activity. Thus, aspects of this
embodiment can include Clostridial toxin light chains comprising an
enzymatic domain having a length of, e.g., at least 350 amino
acids, at least 375 amino acids, at least 400 amino acids, at least
425 amino acids and at least 450 amino acids. Other aspects of this
embodiment can include Clostridial toxin light chains comprising an
enzymatic domain having a length of, e.g., at most 350 amino acids,
at most 375 amino acids, at most 400 amino acids, at most 425 amino
acids and at most 450 amino acids.
[0036] It is also envisioned that any of a variety of Clostridial
toxin H.sub.N regions comprising a translocation domain can be
useful in aspects of the present invention with the proviso that
these active fragments can facilitate the release of the LC from
intracellular vesicles into the cytoplasm of the target cell and
thus participate in executing the overall cellular mechanism
whereby a Clostridial toxin proteolytically cleaves a substrate.
The H.sub.N regions from the heavy chains of Clostridial toxins are
approximately 410-430 amino acids in length and comprise a
translocation domain (Table 1). Research has shown that the entire
length of a H.sub.N region from a Clostridial toxin heavy chain is
not necessary for the translocating activity of the translocation
domain. Thus, aspects of this embodiment can include Clostridial
toxin H.sub.N regions comprising a translocation domain having a
length of, e.g., at least 350 amino acids, at least 375 amino
acids, at least 400 amino acids and at least 425 amino acids. Other
aspects of this embodiment can include Clostridial toxin H.sub.N
regions comprising translocation domain having a length of, e.g.,
at most 350 amino acids, at most 375 amino acids, at most 400 amino
acids and at most 425 amino acids.
[0037] It is also envisioned that any of a variety of Clostridial
toxin H.sub.C regions comprising a binding domain can be useful in
aspects of the present invention with the proviso that these active
fragments can determine the binding activity and binding
specificity of the toxin to the receptor complex located at the
surface of the target cell execute the overall cellular mechanism
whereby a Clostridial toxin proteolytically cleaves a substrate.
The H.sub.C regions from the heavy chains of Clostridial toxins are
approximately 400-440 amino acids in length and comprise a binding
domain (Table 1). Research has shown that the entire length of a
H.sub.C region from a Clostridial toxin heavy chain is not
necessary for the binding activity of the binding domain. Thus,
aspects of this embodiment can include Clostridial toxin H.sub.C
regions comprising a binding domain having a length of, e.g., at
least 350 amino acids, at least 375 amino acids, at least 400 amino
acids and at least 425 amino acids. Other aspects of this
embodiment can include Clostridial toxin H.sub.C regions comprising
a binding domain having a length of, e.g., at most 350 amino acids,
at most 375 amino acids, at most 400 amino acids and at most 425
amino acids.
[0038] Thus, in an embodiment, a Clostridial toxin comprises a
Clostridial toxin enzymatic domain, a Clostridial toxin
translocation domain and a Clostridial toxin binding domain. In an
aspect of this embodiment, a Clostridial toxin comprises a
naturally occurring Clostridial toxin variant, such as, e.g., a
Clostridial toxin isoform or a Clostridial toxin subtype. In
another aspect of this embodiment, a Clostridial toxin comprises a
non-naturally occurring Clostridial toxin variant, such as, e.g., a
conservative Clostridial toxin variant, a non-conservative
Clostridial toxin variant or an active Clostridial toxin fragment,
or any combination thereof. In another aspect of this embodiment, a
Clostridial toxin comprises a Clostridial toxin enzymatic domain or
an active fragment thereof, a Clostridial toxin translocation
domain or an active fragment thereof, a Clostridial toxin binding
domain or an active fragment thereof, or any combination thereof.
In other aspects of this embodiment, a Clostridial toxin can
comprise a BoNT/A, a BoNT/B, a BoNT/C1, a BoNT/D, a BoNT/E, a
BoNT/F, a BoNT/G, a TeNT, a BaNT or a BuNT.
[0039] In another embodiment, a Clostridial toxin comprises a
BoNT/A. In an aspect of this embodiment, a BoNT/A comprises a
BoNT/A enzymatic domain, a BoNT/A translocation domain and a BoNT/A
binding domain. In another aspect of this embodiment, a BoNT/A
comprises SEQ ID NO: 1. In another aspect of this embodiment, a
BoNT/A comprises a naturally occurring BoNT/A variant, such as,
e.g., a BoNT/A isoform or a BoNT/A subtype. In another aspect of
this embodiment, a BoNT/A comprises a naturally occurring BoNT/A
variant of SEQ ID NO: 1, such as, e.g., a BoNT/A isoform of SEQ ID
NO: 1 or a BoNT/A subtype of SEQ ID NO: 1. In still another aspect
of this embodiment, a BoNT/A comprises a non-naturally occurring
BoNT/A variant, such as, e.g., a conservative BoNT/A variant, a
non-conservative BoNT/A variant or an active BoNT/A fragment, or
any combination thereof. In still another aspect of this
embodiment, a BoNT/A comprises a non-naturally occurring BoNT/A
variant of SEQ ID NO: 1, such as, e.g., a conservative BoNT/A
variant of SEQ ID NO: 1, a non-conservative BoNT/A variant of SEQ
ID NO: 1 or an active BoNT/A fragment of SEQ ID NO: 1, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/A comprises a BoNT/A enzymatic domain or an active fragment
thereof, a BoNT/A translocation domain or an active fragment
thereof, a BoNT/A binding domain or an active fragment thereof, or
any combination thereof. In yet another aspect of this embodiment,
a BoNT/A comprising a BoNT/A enzymatic domain of amino acids 1-448
from SEQ ID NO: 1 or an active fragment thereof, a BoNT/A
translocation domain of amino acids 449-871 from SEQ ID NO: 1 or an
active fragment thereof, a BoNT/A binding domain of amino acids
872-1296 from SEQ ID NO: 1 or an active fragment thereof, and any
combination thereof.
[0040] In other aspects of this embodiment, a BoNT/A comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 1, at least 75% amino acid identity with the SEQ ID NO: 1,
at least 80% amino acid identity with SEQ ID NO: 1, at least 85%
amino acid identity with SEQ ID NO: 1, at least 90% amino acid
identity with SEQ ID NO: 1 or at least 95% amino acid identity with
SEQ ID NO: 1. In yet other aspects of this embodiment, a BoNT/A
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 1, at most 75% amino acid identity with
the SEQ ID NO: 1, at most 80% amino acid identity with SEQ ID NO:
1, at most 85% amino acid identity with SEQ ID NO: 1, at most 90%
amino acid identity with SEQ ID NO: 1 or at most 95% amino acid
identity with SEQ ID NO: 1.
[0041] In other aspects of this embodiment, a BoNT/A comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 1.
In other aspects of this embodiment, a BoNT/A comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 1.
In yet other aspects of this embodiment, a BoNT/A comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid deletions relative to SEQ ID NO: 1. In
other aspects of this embodiment, a BoNT/A comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid deletions relative to SEQ ID NO: 1. In still other
aspects of this embodiment, a BoNT/A comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid additions relative to SEQ ID NO: 1. In other aspects of
this embodiment, a BoNT/A comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 1.
[0042] In other aspects of this embodiment, a BoNT/A comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 1. In other aspects
of this embodiment, a BoNT/A comprises a polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10,
20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 1. In yet other aspects of this embodiment,
a BoNT/A comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 1. In other aspects of this embodiment, a BoNT/A comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 1. In still
other aspects of this embodiment, a BoNT/A comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 1. In other aspects of this
embodiment, a BoNT/A comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 contiguous amino acid additions relative to
SEQ ID NO: 1.
[0043] In another embodiment, a Clostridial toxin comprises a
BoNT/B. In an aspect of this embodiment, a BoNT/B comprises a
BoNT/B enzymatic domain, a BoNT/B translocation domain and a BoNT/B
binding domain. In another aspect of this embodiment, a BoNT/B
comprises SEQ ID NO: 2. In another aspect of this embodiment, a
BoNT/B comprises a naturally occurring BoNT/B variant, such as,
e.g., a BoNT/B isoform or a BoNT/B subtype. In another aspect of
this embodiment, a BoNT/B comprises a naturally occurring BoNT/B
variant of SEQ ID NO: 2, such as, e.g., a BoNT/B isoform of SEQ ID
NO: 2 or a BoNT/B subtype of SEQ ID NO: 2. In still another aspect
of this embodiment, a BoNT/B comprises a non-naturally occurring
BoNT/B variant, such as, e.g., a conservative BoNT/B variant, a
non-conservative BoNT/B variant or an active BoNT/B fragment, or
any combination thereof. In still another aspect of this
embodiment, a BoNT/B comprises a non-naturally occurring BoNT/B
variant of SEQ ID NO: 2, such as, e.g., a conservative BoNT/B
variant of SEQ ID NO: 2, a non-conservative BoNT/B variant of SEQ
ID NO: 2 or an active BoNT/B fragment of SEQ ID NO: 2, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/B comprising a BoNT/B enzymatic domain or an active fragment
thereof, a BoNT/B translocation domain or active fragment thereof,
a BoNT/B binding domain or active fragment thereof, and any
combination thereof. In yet another aspect of this embodiment, a
BoNT/B comprising a BoNT/B enzymatic domain of amino acids 1-441
from SEQ ID NO: 2 or active fragment thereof, a BoNT/B
translocation domain of amino acids 442-858 from SEQ ID NO: 2 or
active fragment thereof, a BoNT/B binding domain of amino acids
859-1291 from SEQ ID NO: 2 or active fragment thereof, and any
combination thereof.
[0044] In other aspects of this embodiment, a BoNT/B comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 2, at least 75% amino acid identity with the SEQ ID NO: 2,
at least 80% amino acid identity with SEQ ID NO: 2, at least 85%
amino acid identity with SEQ ID NO: 2, at least 90% amino acid
identity with SEQ ID NO: 2 or at least 95% amino acid identity with
SEQ ID NO: 2. In yet other aspects of this embodiment, a BoNT/B
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 2, at most 75% amino acid identity with
the SEQ ID NO: 2, at most 80% amino acid identity with SEQ ID NO:
2, at most 85% amino acid identity with SEQ ID NO: 2, at most 90%
amino acid identity with SEQ ID NO: 2 or at most 95% amino acid
identity with SEQ ID NO: 2.
[0045] In other aspects of this embodiment, a BoNT/B comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 2.
In other aspects of this embodiment, a BoNT/B comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 2.
In yet other aspects of this embodiment, a BoNT/B comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid deletions relative to SEQ ID NO: 2. In
other aspects of this embodiment, a BoNT/B comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid deletions relative to SEQ ID NO: 2. In still other
aspects of this embodiment, a BoNT/B comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid additions relative to SEQ ID NO: 2. In other aspects of
this embodiment, a BoNT/B comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 2.
[0046] In other aspects of this embodiment, a BoNT/B comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 2. In other aspects
of this embodiment, a BoNT/B comprises a polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10,
20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 2. In yet other aspects of this embodiment,
a BoNT/B comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 2. In other aspects of this embodiment, a BoNT/B comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 2. In still
other aspects of this embodiment, a BoNT/B comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 2. In other aspects of this
embodiment, a BoNT/B comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 contiguous amino acid additions relative to
SEQ ID NO: 2.
[0047] In another embodiment, a Clostridial toxin comprises a
BoNT/C1. In an aspect of this embodiment, a BoNT/C1 comprises a
BoNT/C1 enzymatic domain, a BoNT/C1 translocation domain and a
BoNT/C1 binding domain. In another aspect of this embodiment, a
BoNT/C1 comprises SEQ ID NO: 3. In another aspect of this
embodiment, a BoNT/C1 comprises a naturally occurring BoNT/C1
variant, such as, e.g., a BoNT/C1 isoform or a BoNT/C1 subtype. In
another aspect of this embodiment, a BoNT/C1 comprises a naturally
occurring BoNT/C1 variant of SEQ ID NO: 3, such as, e.g., a BoNT/C1
isoform of SEQ ID NO: 3 or a BoNT/C1 subtype of SEQ ID NO: 3. In
still another aspect of this embodiment, a BoNT/C1 comprises a
non-naturally occurring BoNT/C1 variant, such as, e.g., a
conservative BoNT/C1 variant, a non-conservative BoNT/C1 variant or
an active BoNT/C1 fragment, or any combination thereof. In still
another aspect of this embodiment, a BoNT/C1 comprises a
non-naturally occurring BoNT/C1 variant of SEQ ID NO: 3, such as,
e.g., a conservative BoNT/C1 variant of SEQ ID NO: 3, a
non-conservative BoNT/C1 variant of SEQ ID NO: 3 or an active
BoNT/C1 fragment of SEQ ID NO: 3, or any combination thereof. In
yet another aspect of this embodiment, a BoNT/C1 comprises a
BoNT/C1 enzymatic domain or active fragment thereof, a BoNT/C1
translocation domain or active fragment thereof, a BoNT/C1 binding
domain or active fragment thereof, and any combination thereof. In
yet another aspect of this embodiment, a BoNT/C1 comprises a
BoNT/C1 enzymatic domain of amino acid 1-449 from SEQ ID NO: 3 or
active fragment thereof, a BoNT/C1 translocation domain of amino
acids 450-866 from SEQ ID NO: 3 or active fragment thereof, a
BoNT/C1 binding domain of amino acids 867-1291 from SEQ ID NO: 3 or
active fragment thereof, and any combination thereof.
[0048] In other aspects of this embodiment, a BoNT/C1 comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 3, at least 75% amino acid identity with the SEQ ID NO: 3,
at least 80% amino acid identity with SEQ ID NO: 3, at least 85%
amino acid identity with SEQ ID NO: 3, at least 90% amino acid
identity with SEQ ID NO: 3 or at least 95% amino acid identity with
SEQ ID NO: 3. In yet other aspects of this embodiment, a BoNT/C1
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 3, at most 75% amino acid identity with
the SEQ ID NO: 3, at most 80% amino acid identity with SEQ ID NO:
3, at most 85% amino acid identity with SEQ ID NO: 3, at most 90%
amino acid identity with SEQ ID NO: 3 or at most 95% amino acid
identity with SEQ ID NO: 3.
[0049] In other aspects of this embodiment, a BoNT/C1 comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 3.
In other aspects of this embodiment, a BoNT/C1 comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 3.
In yet other aspects of this embodiment, a BoNT/C1 comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid deletions relative to SEQ ID NO: 3. In
other aspects of this embodiment, a BoNT/C1 comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid deletions relative to SEQ ID NO: 3. In still other
aspects of this embodiment, a BoNT/C1 comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid additions relative to SEQ ID NO: 3. In other aspects of
this embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 3.
[0050] In other aspects of this embodiment, a BoNT/C1 comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 3. In other aspects
of this embodiment, a BoNT/C1 comprises a polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10,
20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 3. In yet other aspects of this embodiment,
a BoNT/C1 comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 3. In other aspects of this embodiment, a BoNT/C1 comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 3. In still
other aspects of this embodiment, a BoNT/C1 comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 3. In other aspects of this
embodiment, a BoNT/C1 comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 contiguous amino acid additions
relative to SEQ ID NO: 3.
[0051] In another embodiment, a Clostridial toxin comprises a
BoNT/D. In an aspect of this embodiment, a BoNT/D comprises a
BoNT/D enzymatic domain, a BoNT/D translocation domain and a BoNT/D
binding domain. In another aspect of this embodiment, a BoNT/D
comprises SEQ ID NO: 4. In another aspect of this embodiment, a
BoNT/D comprises a naturally occurring BoNT/D variant, such as,
e.g., a BoNT/D isoform or a BoNT/D subtype. In another aspect of
this embodiment, a BoNT/D comprises a naturally occurring BoNT/D
variant of SEQ ID NO: 4, such as, e.g., a BoNT/D isoform of SEQ ID
NO: 4 or a BoNT/D subtype of SEQ ID NO: 4. In still another aspect
of this embodiment, a BoNT/D comprises a non-naturally occurring
BoNT/D variant, such as, e.g., a conservative BoNT/D variant, a
non-conservative BoNT/D variant or an active BoNT/D fragment, or
any combination thereof. In still another aspect of this
embodiment, a BoNT/D comprises a non-naturally occurring BoNT/D
variant of SEQ ID NO: 4, such as, e.g., a conservative BoNT/D
variant of SEQ ID NO: 4, a non-conservative BoNT/D variant of SEQ
ID NO: 4 or an active BoNT/D fragment of SEQ ID NO: 4, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/D comprises a BoNT/D enzymatic domain or an active fragment
thereof, a BoNT/D translocation domain or an active fragment
thereof, a BoNT/D binding domain or an active fragment thereof, or
any combination thereof. In yet another aspect of this embodiment,
a BoNT/D comprising a BoNT/D enzymatic domain of amino acids 1-445
from SEQ ID NO: 4 or an active fragment thereof, a BoNT/D
translocation domain of amino acids 446-862 from SEQ ID NO: 4 or an
active fragment thereof, a BoNT/D binding domain of amino acids
863-1276 from SEQ ID NO: 4 or an active fragment thereof, and any
combination thereof.
[0052] In other aspects of this embodiment, a BoNT/D comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 4, at least 75% amino acid identity with the SEQ ID NO: 4,
at least 80% amino acid identity with SEQ ID NO: 4, at least 85%
amino acid identity with SEQ ID NO: 4, at least 90% amino acid
identity with SEQ ID NO: 4 or at least 95% amino acid identity with
SEQ ID NO: 4. In yet other aspects of this embodiment, a BoNT/D
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 4, at most 75% amino acid identity with
the SEQ ID NO: 4, at most 80% amino acid identity with SEQ ID NO:
4, at most 85% amino acid identity with SEQ ID NO: 4, at most 90%
amino acid identity with SEQ ID NO: 4 or at most 95% amino acid
identity with SEQ ID NO: 4.
[0053] In other aspects of this embodiment, a BoNT/D comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 4.
In other aspects of this embodiment, a BoNT/D comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 4.
In yet other aspects of this embodiment, a BoNT/D comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid deletions relative to SEQ ID NO: 4. In
other aspects of this embodiment, a BoNT/D comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid deletions relative to SEQ ID NO: 4. In still other
aspects of this embodiment, a BoNT/D comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid additions relative to SEQ ID NO: 4. In other aspects of
this embodiment, a BoNT/D comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 4.
[0054] In other aspects of this embodiment, a BoNT/D comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 4. In other aspects
of this embodiment, a BoNT/D comprises a polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10,
20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 4. In yet other aspects of this embodiment,
a BoNT/D comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 4. In other aspects of this embodiment, a BoNT/D comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 4. In still
other aspects of this embodiment, a BoNT/D comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 4. In other aspects of this
embodiment, a BoNT/D comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 contiguous amino acid additions relative to
SEQ ID NO: 4.
[0055] In another embodiment, a Clostridial toxin comprises a
BoNT/E. In an aspect of this embodiment, a BoNT/E comprises a
BoNT/E enzymatic domain, a BoNT/E translocation domain and a BoNT/E
binding domain. In another aspect of this embodiment, a BoNT/E
comprises SEQ ID NO: 5. In another aspect of this embodiment, a
BoNT/E comprises a naturally occurring BoNT/E variant, such as,
e.g., a BoNT/E isoform or a BoNT/E subtype. In another aspect of
this embodiment, a BoNT/E comprises a naturally occurring BoNT/E
variant of SEQ ID NO: 5, such as, e.g., a BoNT/E isoform of SEQ ID
NO: 5 or a BoNT/E subtype of SEQ ID NO: 5. In still another aspect
of this embodiment, a BoNT/E comprises a non-naturally occurring
BoNT/E variant, such as, e.g., a conservative BoNT/E variant, a
non-conservative BoNT/E variant or an active BoNT/E fragment, or
any combination thereof. In still another aspect of this
embodiment, a BoNT/E comprises a non-naturally occurring BoNT/E
variant of SEQ ID NO: 5, such as, e.g., a conservative BoNT/E
variant of SEQ ID NO: 5, a non-conservative BoNT/E variant of SEQ
ID NO: 5 or an active BoNT/E fragment of SEQ ID NO: 5, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/E comprising a BoNT/E enzymatic domain or an active fragment
thereof, a BoNT/E translocation domain or active fragment thereof,
a BoNT/E binding domain or active fragment thereof, and any
combination thereof. In yet another aspect of this embodiment, a
BoNT/E comprising a BoNT/E enzymatic domain of amino acids 1-422
from SEQ ID NO: 5 or active fragment thereof, a BoNT/E
translocation domain of amino acids 423-845 from SEQ ID NO: 5 or
active fragment thereof, a BoNT/E binding domain of amino acids
846-1252 from SEQ ID NO: 5 or active fragment thereof, and any
combination thereof.
[0056] In other aspects of this embodiment, a BoNT/E comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 5, at least 75% amino acid identity with the SEQ ID NO: 5,
at least 80% amino acid identity with SEQ ID NO: 5, at least 85%
amino acid identity with SEQ ID NO: 5, at least 90% amino acid
identity with SEQ ID NO: 5 or at least 95% amino acid identity with
SEQ ID NO: 5. In yet other aspects of this embodiment, a BoNT/E
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 5, at most 75% amino acid identity with
the SEQ ID NO: 5, at most 80% amino acid identity with SEQ ID NO:
5, at most 85% amino acid identity with SEQ ID NO: 5, at most 90%
amino acid identity with SEQ ID NO: 5 or at most 95% amino acid
identity with SEQ ID NO: 5.
[0057] In other aspects of this embodiment, a BoNT/E comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 5.
In other aspects of this embodiment, a BoNT/E comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 5.
In yet other aspects of this embodiment, a BoNT/E comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid deletions relative to SEQ ID NO: 5. In
other aspects of this embodiment, a BoNT/E comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid deletions relative to SEQ ID NO: 5. In still other
aspects of this embodiment, a BoNT/E comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid additions relative to SEQ ID NO: 5. In other aspects of
this embodiment, a BoNT/E comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 5.
[0058] In other aspects of this embodiment, a BoNT/E comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 5. In other aspects
of this embodiment, a BoNT/E comprises a polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10,
20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 5. In yet other aspects of this embodiment,
a BoNT/E comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 5. In other aspects of this embodiment, a BoNT/E comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 5. In still
other aspects of this embodiment, a BoNT/E comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 5. In other aspects of this
embodiment, a BoNT/E comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 contiguous amino acid additions relative to
SEQ ID NO: 5.
[0059] In another embodiment, a Clostridial toxin comprises a
BoNT/F. In an aspect of this embodiment, a BoNT/F comprises a
BoNT/F enzymatic domain, a BoNT/F translocation domain and a BoNT/F
binding domain. In another aspect of this embodiment, a BoNT/F
comprises SEQ ID NO: 6. In another aspect of this embodiment, a
BoNT/F comprises a naturally occurring BoNT/F variant, such as,
e.g., a BoNT/F isoform or a BoNT/F subtype. In another aspect of
this embodiment, a BoNT/F comprises a naturally occurring BoNT/F
variant of SEQ ID NO: 6, such as, e.g., a BoNT/F isoform of SEQ ID
NO: 6 or a BoNT/F subtype of SEQ ID NO: 6. In still another aspect
of this embodiment, a BoNT/F comprises a non-naturally occurring
BoNT/F variant, such as, e.g., a conservative BoNT/F variant, a
non-conservative BoNT/F variant or an active BoNT/F fragment, or
any combination thereof. In still another aspect of this
embodiment, a BoNT/F comprises a non-naturally occurring BoNT/F
variant of SEQ ID NO: 6, such as, e.g., a conservative BoNT/F
variant of SEQ ID NO: 6, a non-conservative BoNT/F variant of SEQ
ID NO: 6 or an active BoNT/F fragment of SEQ ID NO: 6, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/F comprises a BoNT/F enzymatic domain or active fragment
thereof, a BoNT/F translocation domain or active fragment thereof,
a BoNT/F binding domain or active fragment thereof, and any
combination thereof. In yet another aspect of this embodiment, a
BoNT/F comprises a BoNT/F enzymatic domain of amino acid 1-439 from
SEQ ID NO: 6 or active fragment thereof, a BoNT/F translocation
domain of amino acids 440-864 from SEQ ID NO: 6 or active fragment
thereof, a BoNT/F binding domain of amino acids 865-1274 from SEQ
ID NO: 6 or active fragment thereof, and any combination
thereof.
[0060] In other aspects of this embodiment, a BoNT/F comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 6, at least 75% amino acid identity with the SEQ ID NO: 6,
at least 80% amino acid identity with SEQ ID NO: 6, at least 85%
amino acid identity with SEQ ID NO: 6, at least 90% amino acid
identity with SEQ ID NO: 6 or at least 95% amino acid identity with
SEQ ID NO: 6. In yet other aspects of this embodiment, a BoNT/F
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 6, at most 75% amino acid identity with
the SEQ ID NO: 6, at most 80% amino acid identity with SEQ ID NO:
6, at most 85% amino acid identity with SEQ ID NO: 6, at most 90%
amino acid identity with SEQ ID NO: 6 or at most 95% amino acid
identity with SEQ ID NO: 6.
[0061] In other aspects of this embodiment, a BoNT/F comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 6.
In other aspects of this embodiment, a BoNT/F comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 6.
In yet other aspects of this embodiment, a BoNT/F comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid deletions relative to SEQ ID NO: 6. In
other aspects of this embodiment, a BoNT/F comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid deletions relative to SEQ ID NO: 6. In still other
aspects of this embodiment, a BoNT/F comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid additions relative to SEQ ID NO: 6. In other aspects of
this embodiment, a BoNT/F comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 6.
[0062] In other aspects of this embodiment, a BoNT/F comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 6. In other aspects
of this embodiment, a BoNT/F comprises a polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10,
20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 6. In yet other aspects of this embodiment,
a BoNT/F comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 6. In other aspects of this embodiment, a BoNT/F comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 6. In still
other aspects of this embodiment, a BoNT/F comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 6. In other aspects of this
embodiment, a BoNT/F comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 contiguous amino acid additions relative to
SEQ ID NO: 6.
[0063] In another embodiment, a Clostridial toxin comprises a
BoNT/G. In an aspect of this embodiment, a BoNT/G comprises a
BoNT/G enzymatic domain, a BoNT/G translocation domain and a BoNT/G
binding domain. In another aspect of this embodiment, a BoNT/G
comprises SEQ ID NO: 7. In another aspect of this embodiment, a
BoNT/G comprises a naturally occurring BoNT/G variant, such as,
e.g., a BoNT/G isoform or a BoNT/G subtype. In another aspect of
this embodiment, a BoNT/G comprises a naturally occurring BoNT/G
variant of SEQ ID NO: 7, such as, e.g., a BoNT/G isoform of SEQ ID
NO: 7 or a BoNT/G subtype of SEQ ID NO: 7. In still another aspect
of this embodiment, a BoNT/G comprises a non-naturally occurring
BoNT/G variant, such as, e.g., a conservative BoNT/G variant, a
non-conservative BoNT/G variant or an active BoNT/G fragment, or
any combination thereof. In still another aspect of this
embodiment, a BoNT/D comprises a non-naturally occurring BoNT/G
variant of SEQ ID NO: 7, such as, e.g., a conservative BoNT/G
variant of SEQ ID NO: 7, a non-conservative BoNT/G variant of SEQ
ID NO: 7 or an active BoNT/G fragment of SEQ ID NO: 7, or any
combination thereof. In yet another aspect of this embodiment, a
BoNT/G comprises a BoNT/G enzymatic domain or an active fragment
thereof, a BoNT/G translocation domain or an active fragment
thereof, a BoNT/G binding domain or an active fragment thereof, or
any combination thereof. In yet another aspect of this embodiment,
a BoNT/G comprising a BoNT/G enzymatic domain of amino acids 1-446
from SEQ ID NO: 7 or an active fragment thereof, a BoNT/G
translocation domain of amino acids 447-863 from SEQ ID NO: 7 or an
active fragment thereof, a BoNT/G binding domain of amino acids
864-1297 from SEQ ID NO: 7 or an active fragment thereof, and any
combination thereof.
[0064] In other aspects of this embodiment, a BoNT/G comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 7, at least 75% amino acid identity with the SEQ ID NO: 7,
at least 80% amino acid identity with SEQ ID NO: 7, at least 85%
amino acid identity with SEQ ID NO: 7, at least 90% amino acid
identity with SEQ ID NO: 7 or at least 95% amino acid identity with
SEQ ID NO: 7. In yet other aspects of this embodiment, a BoNT/G
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 7, at most 75% amino acid identity with
the SEQ ID NO: 7, at most 80% amino acid identity with SEQ ID NO:
7, at most 85% amino acid identity with SEQ ID NO: 7, at most 90%
amino acid identity with SEQ ID NO: 7 or at most 95% amino acid
identity with SEQ ID NO: 7.
[0065] In other aspects of this embodiment, a BoNT/G comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 7.
In other aspects of this embodiment, a BoNT/G comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 7.
In yet other aspects of this embodiment, a BoNT/G comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid deletions relative to SEQ ID NO: 7. In
other aspects of this embodiment, a BoNT/G comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid deletions relative to SEQ ID NO: 7. In still other
aspects of this embodiment, a BoNT/G comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid additions relative to SEQ ID NO: 7. In other aspects of
this embodiment, a BoNT/G comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 7.
[0066] In other aspects of this embodiment, a BoNT/G comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 7. In other aspects
of this embodiment, a BoNT/G comprises a polypeptide having, e.g.,
at least one, two, three, four, five, six, seven, eight, nine, 10,
20, 30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 7. In yet other aspects of this embodiment,
a BoNT/G comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 7. In other aspects of this embodiment, a BoNT/G comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 7. In still
other aspects of this embodiment, a BoNT/G comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 7. In other aspects of this
embodiment, a BoNT/G comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 contiguous amino acid additions relative to
SEQ ID NO: 7.
[0067] In another embodiment, a Clostridial toxin comprises a TeNT.
In an aspect of this embodiment, a TeNT comprises a TeNT enzymatic
domain, a TeNT translocation domain and a TeNT binding domain. In
an aspect of this embodiment, a TeNT comprises SEQ ID NO: 8. In
another aspect of this embodiment, a TeNT comprises a naturally
occurring TeNT variant, such as, e.g., a TeNT isoform or a TeNT
subtype. In another aspect of this embodiment, a TeNT comprises a
naturally occurring TeNT variant of SEQ ID NO: 8, such as, e.g., a
TeNT isoform of SEQ ID NO: 8 or a TeNT subtype of SEQ ID NO: 8. In
still another aspect of this embodiment, a TeNT comprises a
non-naturally occurring TeNT variant, such as, e.g., a conservative
TeNT variant, a non-conservative TeNT variant or an active TeNT
fragment, or any combination thereof. In still another aspect of
this embodiment, a TeNT comprises a non-naturally occurring TeNT
variant of SEQ ID NO: 8, such as, e.g., a conservative TeNT variant
of SEQ ID NO: 8, a non-conservative TeNT variant of SEQ ID NO: 8 or
an active TeNT fragment of SEQ ID NO: 8, or any combination
thereof. In yet another aspect of this embodiment, a TeNT
comprising a TeNT enzymatic domain or an active fragment thereof, a
TeNT translocation domain or active fragment thereof, a TeNT
binding domain or active fragment thereof, and any combination
thereof. In yet another aspect of this embodiment, a TeNT
comprising a TeNT enzymatic domain of amino acids 1-457 from SEQ ID
NO: 8 or active fragment thereof, a TeNT translocation domain of
amino acids 458-879 from SEQ ID NO: 8 or active fragment thereof, a
TeNT binding domain of amino acids 880-1315 from SEQ ID NO: 8 or
active fragment thereof, and any combination thereof.
[0068] In other aspects of this embodiment, a TeNT comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 8, at least 75% amino acid identity with the SEQ ID NO: 8,
at least 80% amino acid identity with SEQ ID NO: 8, at least 85%
amino acid identity with SEQ ID NO: 8, at least 90% amino acid
identity with SEQ ID NO: 8 or at least 95% amino acid identity with
SEQ ID NO: 8. In yet other aspects of this embodiment, a TeNT
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 8, at most 75% amino acid identity with
the SEQ ID NO: 8, at most 80% amino acid identity with SEQ ID NO:
8, at most 85% amino acid identity with SEQ ID NO: 8, at most 90%
amino acid identity with SEQ ID NO: 8 or at most 95% amino acid
identity with SEQ ID NO: 8.
[0069] In other aspects of this embodiment, a TeNT comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 8.
In other aspects of this embodiment, a TeNT comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid substitutions relative to SEQ ID NO: 8. In yet other
aspects of this embodiment, a TeNT comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid
deletions relative to SEQ ID NO: 8. In other aspects of this
embodiment, a TeNT comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 non-contiguous amino acid deletions
relative to SEQ ID NO: 8. In still other aspects of this
embodiment, a TeNT comprises a polypeptide having, e.g., at most
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 8. In other aspects of this embodiment, a
TeNT comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 non-contiguous amino acid additions relative to SEQ
ID NO: 8.
[0070] In other aspects of this embodiment, a TeNT comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 8. In other aspects
of this embodiment, a TeNT comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 8. In yet other aspects of this embodiment,
a TeNT comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 8. In other aspects of this embodiment, a TeNT comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 8. In still
other aspects of this embodiment, a TeNT comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 8. In other aspects of this
embodiment, a TeNT comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 contiguous amino acid additions relative to
SEQ ID NO: 8.
[0071] In another embodiment, a Clostridial toxin comprises a BaNT.
In an aspect of this embodiment, a BaNT comprises a BaNT enzymatic
domain, a BaNT translocation domain and a BaNT binding domain. In
another aspect of this embodiment, a BaNT comprises SEQ ID NO: 9.
In another aspect of this embodiment, a BaNT comprises a naturally
occurring BaNT variant, such as, e.g., a BaNT isoform or a BaNT
subtype. In another aspect of this embodiment, a BaNT comprises a
naturally occurring BaNT variant of SEQ ID NO: 9, such as, e.g., a
BaNT isoform of SEQ ID NO: 9 or a BaNT subtype of SEQ ID NO: 9. In
still another aspect of this embodiment, a BaNT comprises a
non-naturally occurring BaNT variant, such as, e.g., a conservative
BaNT variant, a non-conservative BaNT variant or an active BaNT
fragment, or any combination thereof. In still another aspect of
this embodiment, a BaNT comprises a non-naturally occurring BaNT
variant of SEQ ID NO: 9, such as, e.g., a conservative BaNT variant
of SEQ ID NO: 9, a non-conservative BaNT variant of SEQ ID NO: 9 or
an active BaNT fragment of SEQ ID NO: 9, or any combination
thereof. In yet another aspect of this embodiment, a BaNT comprises
a BaNT enzymatic domain or an active fragment thereof, a BaNT
translocation domain or an active fragment thereof, a BaNT binding
domain or an active fragment thereof, or any combination thereof.
In yet another aspect of this embodiment, a BaNT comprising a BaNT
enzymatic domain of amino acids 1-448 from SEQ ID NO: 9 or an
active fragment thereof, a BaNT translocation domain of amino acids
449-871 from SEQ ID NO: 9 or an active fragment thereof, a BaNT
binding domain of amino acids 872-1296 from SEQ ID NO: 9 or an
active fragment thereof, and any combination thereof.
[0072] In other aspects of this embodiment, a BaNT comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 9, at least 75% amino acid identity with the SEQ ID NO: 9,
at least 80% amino acid identity with SEQ ID NO: 9, at least 85%
amino acid identity with SEQ ID NO: 9, at least 90% amino acid
identity with SEQ ID NO: 9 or at least 95% amino acid identity with
SEQ ID NO: 9. In yet other aspects of this embodiment, a BaNT
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 9, at most 75% amino acid identity with
the SEQ ID NO: 9, at most 80% amino acid identity with SEQ ID NO:
9, at most 85% amino acid identity with SEQ ID NO: 9, at most 90%
amino acid identity with SEQ ID NO: 9 or at most 95% amino acid
identity with SEQ ID NO: 9.
[0073] In other aspects of this embodiment, a BaNT comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 9.
In other aspects of this embodiment, a BaNT comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid substitutions relative to SEQ ID NO: 9. In yet other
aspects of this embodiment, a BaNT comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid
deletions relative to SEQ ID NO: 9. In other aspects of this
embodiment, a BaNT comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 non-contiguous amino acid deletions
relative to SEQ ID NO: 9. In still other aspects of this
embodiment, a BaNT comprises a polypeptide having, e.g., at most
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 9. In other aspects of this embodiment, a
BaNT comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 non-contiguous amino acid additions relative to SEQ
ID NO: 9.
[0074] In other aspects of this embodiment, a BaNT comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 9. In other aspects
of this embodiment, a BaNT comprises a polypeptide having, e.g., at
least one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 contiguous amino acid substitutions
relative to SEQ ID NO: 9. In yet other aspects of this embodiment,
a BaNT comprises a polypeptide having, e.g., at most one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 9. In other aspects of this embodiment, a BaNT comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid deletions relative to SEQ ID NO: 9. In still
other aspects of this embodiment, a BaNT comprises a polypeptide
having, e.g., at most one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino
acid additions relative to SEQ ID NO: 9. In other aspects of this
embodiment, a BaNT comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 contiguous amino acid additions relative to
SEQ ID NO: 9.
[0075] In another embodiment, a Clostridial toxin comprises a BuNT.
In an aspect of this embodiment, a BuNT comprises a BuNT enzymatic
domain, a BuNT translocation domain and a BuNT binding domain. In
another aspect of this embodiment, a BuNT comprises SEQ ID NO: 10.
In another aspect of this embodiment, a BuNT comprises a naturally
occurring BuNT variant, such as, e.g., a BuNT isoform or a BuNT
subtype. In another aspect of this embodiment, a BuNT comprises a
naturally occurring BuNT variant of SEQ ID NO: 10, such as, e.g., a
BuNT isoform of SEQ ID NO: 10 or a BuNT subtype of SEQ ID NO: 10.
In still another aspect of this embodiment, a BuNT comprises a
non-naturally occurring BuNT variant, such as, e.g., a conservative
BuNT variant, a non-conservative BuNT variant or an active BuNT
fragment, or any combination thereof. In still another aspect of
this embodiment, a BuNT comprises a non-naturally occurring BuNT
variant of SEQ ID NO: 10, such as, e.g., a conservative BuNT
variant of SEQ ID NO: 10, a non-conservative BuNT variant of SEQ ID
NO: 10 or an active BuNT fragment of SEQ ID NO: 10, or any
combination thereof. In yet another aspect of this embodiment, a
BuNT comprises a BuNT enzymatic domain or an active fragment
thereof, a BuNT translocation domain or an active fragment thereof,
a BuNT binding domain or an active fragment thereof, or any
combination thereof. In yet another aspect of this embodiment, a
BuNT comprising a BuNT enzymatic domain of amino acids 1-448 from
SEQ ID NO: 10 or an active fragment thereof, a BuNT translocation
domain of amino acids 449-871 from SEQ ID NO: 10 or an active
fragment thereof, a BuNT binding domain of amino acids 872-1296
from SEQ ID NO: 10 or an active fragment thereof, and any
combination thereof.
[0076] In other aspects of this embodiment, a BuNT comprises a
polypeptide having, e.g., at least 70% amino acid identity with SEQ
ID NO: 10, at least 75% amino acid identity with the SEQ ID NO: 10,
at least 80% amino acid identity with SEQ ID NO: 10, at least 85%
amino acid identity with SEQ ID NO: 10, at least 90% amino acid
identity with SEQ ID NO: 10 or at least 95% amino acid identity
with SEQ ID NO: 10. In yet other aspects of this embodiment, a BuNT
comprises a polypeptide having, e.g., at most 70% amino acid
identity with SEQ ID NO: 10, at most 75% amino acid identity with
the SEQ ID NO: 10, at most 80% amino acid identity with SEQ ID NO:
10, at most 85% amino acid identity with SEQ ID NO: 10, at most 90%
amino acid identity with SEQ ID NO: 10 or at most 95% amino acid
identity with SEQ ID NO: 10.
[0077] In other aspects of this embodiment, a BuNT comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
non-contiguous amino acid substitutions relative to SEQ ID NO: 10.
In other aspects of this embodiment, a BuNT comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous
amino acid substitutions relative to SEQ ID NO: 10. In yet other
aspects of this embodiment, a BuNT comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, 100, 200 or 500 non-contiguous amino acid
deletions relative to SEQ ID NO: 10. In other aspects of this
embodiment, a BuNT comprises a polypeptide having, e.g., at least
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 non-contiguous amino acid deletions
relative to SEQ ID NO: 10. In still other aspects of this
embodiment, a BuNT comprises a polypeptide having, e.g., at most
one, two, three, four, five, six, seven, eight, nine, 10, 20, 30,
40, 50, 100, 200 or 500 non-contiguous amino acid additions
relative to SEQ ID NO: 10. In other aspects of this embodiment, a
BuNT comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 non-contiguous amino acid additions relative to SEQ
ID NO: 10.
[0078] In other aspects of this embodiment, a BuNT comprises a
polypeptide having, e.g., at most one, two, three, four, five, six,
seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous
amino acid substitutions relative to SEQ ID NO: 10. In other
aspects of this embodiment, a BuNT comprises a polypeptide having,
e.g., at least one, two, three, four, five, six, seven, eight,
nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid
substitutions relative to SEQ ID NO: 10. In yet other aspects of
this embodiment, a BuNT comprises a polypeptide having, e.g., at
most one, two, three, four, five, six, seven, eight, nine, 10, 20,
30, 40, 50, 100, 200 or 500 contiguous amino acid deletions
relative to SEQ ID NO: 10. In other aspects of this embodiment, a
BuNT comprises a polypeptide having, e.g., at least one, two,
three, four, five, six, seven, eight, nine, 10, 20, 30, 40, 50,
100, 200 or 500 contiguous amino acid deletions relative to SEQ ID
NO: 10. In still other aspects of this embodiment, a BuNT comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, 100, 200 or 500
contiguous amino acid additions relative to SEQ ID NO: 10. In other
aspects of this embodiment, a BuNT comprises a polypeptide having,
e.g., at least one, two, three, four, five, six, seven, eight,
nine, 10, 20, 30, 40, 50, 100, 200 or 500 contiguous amino acid
additions relative to SEQ ID NO: 10.
[0079] As mentioned above, a Clostridial toxin is converted from a
single polypeptide form into a di-chain molecule by proteolytic
cleavage. The location of the di-chain loop protease cleavage site
for Clostridial toxins is shown (Table 2). Cleavage within the
di-chain loop does not appear to be confined to a single peptide
bond. Thus, cleavage of a Clostridial toxin with a
naturally-occurring di-chain loop protease results in the lost of
several residues centered around the original cleavage site. This
loss is limited to a few amino acids located between the two
cysteine residues that form the disulfide bridge. As a non-limiting
example, BoNT/A single-chain polypeptide cleavage ultimately
results in the loss of a ten amino acids within the di-chain loop.
For BoNTs, cleavage at K448-A449 converts the single-chain form of
BoNT/A into the di-chain form; cleavage at K441-A442 converts the
single-chain form of BoNT/B into the di-chain form; cleavage at
K449-T450 converts the single-chain form of BoNT/C1 into the
di-chain form; cleavage at R445-D446 converts the single-chain form
of BoNT/D into the di-chain form; cleavage at R422-K423 converts
the single-chain form of BoNT/E into the di-chain form; cleavage at
K439-A440 converts the single-chain form of BoNT/F into the
di-chain form; and cleavage at K446-5447 converts the single-chain
form of BoNT/G into the di-chain form. Proteolytic cleavage of the
single-chain form of TeNT at of A457-5458 results in the di-chain
form. Proteolytic cleavage of the single-chain form of BaNT at of
K431-N432 results in the di-chain form. Proteolytic cleavage of the
single-chain form of BuNT at of R422-K423 results in the di-chain
form.
TABLE-US-00002 TABLE 2 Di-chain Loop Region of Clostidial Toxins
Toxin SEQ ID NO: Di-Chain Loop Region Including a Di-Chain Protease
Cleavage Site BoNT/A 11 CVRGIITSKTKSLDKGYNK*----ALNDLC BoNT/B 12
CKSVK*-------------------APGIC BoNT/C1 13
CHKAIDGRSLYNK*------------TLDC BoNT/D 14
CLRLTKNSR*---------------DDSTC BoNT/E 15
CKNIVSVKGIR*--------------KSIC BoNT/F 16
CKSVIPRKGTK*------------APPRLC BoNT/G 17
CKPVMYKNTGK*--------------SEQC TeNT 18
CKKIIPPTNIRENLYNRTA*SLTDLGGELC BaNT 19
CKSIVSKKGTK*--------------NSLC BuNT 20
CKNIVSVKGIR*--------------KSIC The amino acid sequence displayed
are as follows: BoNT/A, residues 430-454 of SEQ ID NO: 1; BoNT/B,
residues 437-446 of SEQ ID NO: 2; BoNT/C1, residues 437-453 of SEQ
ID NO: 3; BoNT/D, residues 437-450 of SEQ ID NO: 4; BoNT/E,
residues 412-426 of SEQ ID NO: 5; BoNT/F, residues 429-445 of SEQ
ID NO: 6; BoNT/G, residues 436-450 of SEQ ID NO: 7; TeNT, residues
439-467 of SEQ ID NO: 8; BaNT, residues 421-435 of SEQ ID NO: 9;
and BuNT, residues 412-426 of SEQ ID NO: 10. An asterisks (*)
indicates the peptide bond of the P.sub.1-P.sub.1' cleavage site
that is believed to be cleaved by a Clostridial toxin di-chain loop
protease.
[0080] However, it should also be noted that additional cleavage
sites within the di-chain loop also appear to be cleaved resulting
in the generation of a small peptide fragment being lost. As a
non-limiting example, BoNT/A single-chain polypeptide cleavage
ultimately results in the loss of a ten amino acid fragment within
the di-chain loop. Thus, cleavage at S441-L442 converts the single
polypeptide form of BoNT/A into the di-chain form; cleavage at
G444-1445 converts the single polypeptide form of BoNT/B into the
di-chain form; cleavage at S445-L446 converts the single
polypeptide form of BoNT/C1 into the di-chain form; cleavage at
K442-N443 converts the single polypeptide form of BoNT/D into the
di-chain form; cleavage at K419-G420 converts the single
polypeptide form of BoNT/E into the di-chain form; cleavage at
K423-S424 converts the single polypeptide form of BoNT/E into the
di-chain form; cleavage at K436-G437 converts the single
polypeptide form of BoNT/F into the di-chain form; cleavage at
T444-G445 converts the single polypeptide form of BoNT/G into the
di-chain form; and cleavage at E448-Q449 converts the single
polypeptide form of BoNT/G into the di-chain form.
[0081] Aspects of the present invention provide, in part, a
Clostridial toxin di-chain loop region. As used herein, the term
"Clostridial toxin di-chain loop region" means the loop region of a
Clostridial toxin formed by a disulfide bridge located between the
LC domain and the HC domain of a naturally-occurring Clostridial
toxin. A Clostridial toxin di-chain loop region includes, without
limitation, a BoNT/A di-chain loop region, a BoNT/B di-chain loop
region, a BoNT/C1 di-chain loop region, a BoNT/D di-chain loop
region, a BoNT/E di-chain loop region, a BoNT/F di-chain loop
region, a BoNT/G di-chain loop region, a TeNT di-chain loop region,
a BaNT di-chain loop region, and a BuNT di-chain loop region. A
non-limiting example of a BoNT/A di-chain loop region is amino acid
sequence CVRGIITSKTKSLDKGYNKALNDLC (SEQ ID NO: 11). A non-limiting
example of a BoNT/B di-chain loop region is the amino acid sequence
CKSVKAPGIC (SEQ ID NO: 12). A non-limiting example of a BoNT/C1
di-chain loop region is the amino acid sequence CHKAIDGRSLYNKTLDC
(SEQ ID NO: 13). A non-limiting example of a BoNT/D di-chain loop
region is the amino acid sequence CLRLTKNSRDDSTC (SEQ ID NO: 14). A
non-limiting example of a BoNT/E di-chain loop region is the amino
acid sequence CKNIVSVKGIRKSIC (SEQ ID NO: 15). A non-limiting
example of a BoNT/F di-chain loop region is the amino acid sequence
CKSVIPRKGTKAPPRLC (SEQ ID NO: 16). A non-limiting example of a
BoNT/G di-chain loop region is the amino acid sequence
CKPVMYKNTGKSEQC (SEQ ID NO: 17). A non-limiting example of a TeNT
di-chain loop region is the amino acid sequence
CKKIIPPTNIRENLYNRTASLTDLGGELC (SEQ ID NO: 18). A non-limiting
example of a BaNT di-chain loop region is the amino acid sequence
CKSIVSKKGTKNSLC (SEQ ID NO: 19). A non-limiting example of a BuNT
di-chain loop region is the amino acid sequence CKNIVSVKGIRKSIC
(SEQ ID NO: 20). As discussed below, SEQ ID NO: 11 through SEQ ID
NO: 20 can serve as reference Clostridial toxin di-chain loop
region sequences.
[0082] A Clostridial toxin di-chain loop region useful in aspects
of the invention includes, without limitation, naturally occurring
Clostridial toxin di-chain loop region; naturally occurring
Clostridial toxin di-chain loop region variants; and
non-naturally-occurring Clostridial toxin di-chain loop region
variants, such as, e.g., conservative Clostridial toxin di-chain
loop region variants, non-conservative Clostridial toxin di-chain
loop region variants and Clostridial toxin di-chain loop region
peptidomimetics. As used herein, the term "Clostridial toxin
di-chain loop region variant," whether naturally-occurring or
non-naturally-occurring, means a Clostridial toxin di-chain loop
region that has at least one amino acid change from the
corresponding region of the disclosed reference sequences and can
be described in percent identity to the corresponding region of
that reference sequence. Any of a variety of sequence alignment
methods can be used to determine percent identity, including,
without limitation, global methods, local methods and hybrid
methods, such as, e.g., segment approach methods. Protocols to
determine percent identity are routine procedures within the scope
of one skilled in the art and from the teaching herein.
[0083] As used herein, the term "naturally occurring Clostridial
toxin di-chain loop region variant" means any Clostridial toxin
di-chain loop region produced without the aid of any human
manipulation, including, without limitation, Clostridial toxin
di-chain loop region isoforms produced from alternatively-spliced
transcripts, Clostridial toxin di-chain loop region isoforms
produced by spontaneous mutation and Clostridial toxin di-chain
loop region subtypes. Non-limiting examples of a Clostridial toxin
di-chain loop region isoform include, e.g., BoNT/A di-chain loop
region isoforms, BoNT/B di-chain loop region isoforms, BoNT/C1
di-chain loop region isoforms, BoNT/D di-chain loop region
isoforms, BoNT/E di-chain loop region isoforms, BoNT/F di-chain
loop region isoforms, BoNT/G di-chain loop region isoforms, TeNT
di-chain loop region isoforms, BaNT di-chain loop region isoforms,
and BuNT di-chain loop region isoforms. Non-limiting examples of a
Clostridial toxin subtype include, e.g., BoNT/A di-chain loop
region subtypes such as, e.g., a BoNT/A1 di-chain loop region, a
BoNT/A2 di-chain loop region, a BoNT/A3 di-chain loop region and a
BoNT/A4 di-chain loop region; BoNT/B di-chain loop region subtypes,
such as, e.g., a BoNT/B1 di-chain loop region, a BoNT/B2 di-chain
loop region, a BoNT/B bivalent di-chain loop region and a BoNT/B
nonproteolytic di-chain loop region; BoNT/C1 di-chain loop region
subtypes, such as, e.g., a BoNT/C1-1 di-chain loop region and a
BoNT/C1-2 di-chain loop region; BoNT/E di-chain loop region
subtypes, such as, e.g., a BoNT/E1 di-chain loop region, a BoNT/E2
di-chain loop region and a BoNT/E3 di-chain loop region; and BoNT/F
di-chain loop region subtypes, such as, e.g., a BoNT/F1 di-chain
loop region, a BoNT/F2 di-chain loop region, a BoNT/F3 di-chain
loop region and a BoNT/F4 di-chain loop region.
[0084] As used herein, the term "non-naturally occurring
Clostridial toxin di-chain loop region variant" means any
Clostridial toxin di-chain loop region produced with the aid of
human manipulation, including, without limitation, Clostridial
toxin di-chain loop region variants produced by genetic engineering
using random mutagenesis or rational design and Clostridial toxin
di-chain loop region variants produced by chemical synthesis.
Non-limiting examples of non-naturally occurring Clostridial toxin
di-chain loop region variants include, e.g., conservative
Clostridial toxin di-chain loop region variants, non-conservative
Clostridial toxin di-chain loop region variants and Clostridial
toxin di-chain loop region peptidomimetics.
[0085] As used herein, the term "conservative Clostridial toxin
di-chain loop region variant" means a Clostridial toxin di-chain
loop region that has at least one amino acid substituted by another
amino acid or an amino acid analog that has at least one property
similar to that of the original amino acid from the reference
Clostridial toxin di-chain loop region sequence. Examples of
properties include, without limitation, similar size, topography,
charge, hydrophobicity, hydrophilicity, lipophilicity,
covalent-bonding capacity, hydrogen-bonding capacity, a
physicochemical property, of the like, or any combination thereof.
A conservative Clostridial toxin di-chain loop region variant can
function in substantially the same manner as the reference
Clostridial toxin di-chain loop region on which the conservative
Clostridial toxin di-chain loop region variant is based, and can be
substituted for the reference Clostridial toxin di-chain loop
region in any aspect of the present invention. A conservative
Clostridial toxin di-chain loop region variant may substitute one
or more amino acids, two or more amino acids, three or more amino
acids, four or more amino acids or five or more amino acids from
the reference Clostridial toxin di-chain loop region on which the
conservative Clostridial toxin di-chain loop region variant is
based. A conservative Clostridial toxin di-chain loop region
variant can also possess at least 50% amino acid identity, 65%
amino acid identity, 75% amino acid identity, 85% amino acid
identity or 95% amino acid identity to the reference Clostridial
toxin di-chain loop region on which the conservative Clostridial
toxin di-chain loop region variant is based. Non-limiting examples
of a conservative Clostridial toxin di-chain loop region variant
include, e.g., conservative BoNT/A di-chain loop region variants,
conservative BoNT/B di-chain loop region variants, conservative
BoNT/C1 di-chain loop region variants, conservative BoNT/D di-chain
loop region variants, conservative BoNT/E di-chain loop region
variants, conservative BoNT/F di-chain loop region variants,
conservative BoNT/G di-chain loop region variants, conservative
TeNT di-chain loop region variants, conservative BaNT di-chain loop
region variants and conservative BuNT di-chain loop region
variants.
[0086] As used herein, the term "non-conservative Clostridial toxin
di-chain loop region variant" means a Clostridial toxin di-chain
loop region in which 1) at least one amino acid is deleted from the
reference Clostridial toxin di-chain loop region on which the
non-conservative Clostridial toxin di-chain loop region variant is
based; 2) at least one amino acid added to the reference
Clostridial toxin di-chain loop region on which the
non-conservative Clostridial toxin di-chain loop region is based;
or 3) at least one amino acid is substituted by another amino acid
or an amino acid analog that does not share any property similar to
that of the original amino acid from the reference Clostridial
toxin di-chain loop region sequence. A non-conservative Clostridial
toxin di-chain loop region variant can function in substantially
the same manner as the reference Clostridial toxin di-chain loop
region on which the non-conservative Clostridial toxin di-chain
loop region is based, and can be substituted for the reference
Clostridial toxin di-chain loop region in any aspect of the present
invention. A non-conservative Clostridial toxin di-chain loop
region variant can add one or more amino acids, two or more amino
acids, three or more amino acids, four or more amino acids, five or
more amino acids, and ten or more amino acids to the reference
Clostridial toxin di-chain loop region on which the
non-conservative Clostridial toxin di-chain loop region variant is
based. A non-conservative Clostridial toxin di-chain loop region
may substitute one or more amino acids, two or more amino acids,
three or more amino acids, four or more amino acids or five or more
amino acids from the reference Clostridial toxin di-chain loop
region on which the non-conservative Clostridial toxin di-chain
loop region variant is based. A non-conservative Clostridial toxin
di-chain loop region variant can also possess at least 50% amino
acid identity, 65% amino acid identity, 75% amino acid identity,
85% amino acid identity or 95% amino acid identity to the reference
Clostridial toxin di-chain loop region on which the
non-conservative Clostridial toxin di-chain loop region variant is
based. Non-limiting examples of a non-conservative Clostridial
toxin di-chain loop region variant include, e.g., non-conservative
BoNT/A di-chain loop region variants, non-conservative BoNT/B
di-chain loop region variants, non-conservative BoNT/C1 di-chain
loop region variants, non-conservative BoNT/D di-chain loop region
variants, non-conservative BoNT/E di-chain loop region variants,
non-conservative BoNT/F di-chain loop region variants,
non-conservative BoNT/G di-chain loop region variants,
non-conservative TeNT di-chain loop region variants,
non-conservative BaNT di-chain loop region variants and
non-conservative BuNT di-chain loop region variants.
[0087] As used herein, the term "Clostridial toxin di-chain loop
region peptidomimetic" means a Clostridial toxin di-chain loop
region that has at least one amino acid substituted by a
non-natural oligomer that has at least one property similar to that
of the first amino acid. Examples of properties include, without
limitation, topography of a peptide primary structural element,
functionality of a peptide primary structural element, topology of
a peptide secondary structural element, functionality of a peptide
secondary structural element, of the like, or any combination
thereof. A Clostridial toxin di-chain loop region peptidomimetic
can function in substantially the same manner as the reference
Clostridial toxin di-chain loop region on which the Clostridial
toxin di-chain loop region peptidomimetic is based, and can be
substituted for the reference Clostridial toxin di-chain loop
region in any aspect of the present invention. A Clostridial toxin
di-chain loop region peptidomimetic may substitute one or more
amino acids, two or more amino acids, three or more amino acids,
four or more amino acids or five or more amino acids from the
reference Clostridial toxin di-chain loop region on which the
Clostridial toxin di-chain loop region peptidomimetic is based. A
Clostridial toxin di-chain loop region peptidomimetic can also
possess at least 50% amino acid identity, at least 65% amino acid
identity, at least 75% amino acid identity, at least 85% amino acid
identity or at least 95% amino acid identity to the reference
Clostridial toxin di-chain loop region on which the Clostridial
toxin di-chain loop region peptidomimetic is based. For examples of
peptidomimetic methods see, e.g., Amy S. Ripka & Daniel H.
Rich, Peptidomimetic design, 2(4) CURR. OPIN. CHEM. BIOL. 441-452
(1998); and M. Angels Estiarte & Daniel H. Rich,
Peptidomimetics for Drug Design, 803-861 (BURGER'S MEDICINAL
CHEMISTRY AND DRUG DISCOVERY VOl. 1 PRINCIPLE AND PRACTICE, Donald
J. Abraham ed., Wiley-Interscience, 6.sup.th ed 2003). Non-limiting
examples of a Clostridial toxin di-chain loop region peptidomimetic
include, e.g., BoNT/A di-chain loop region peptidomimetics, BoNT/B
di-chain loop region peptidomimetics, BoNT/C1 di-chain loop region
peptidomimetics, BoNT/D di-chain loop region peptidomimetics,
BoNT/E di-chain loop region peptidomimetics, BoNT/F di-chain loop
region peptidomimetics, BoNT/G di-chain loop region
peptidomimetics, TeNT di-chain loop region peptidomimetics, BaNT
di-chain loop region peptidomimetics and BuNT di-chain loop region
peptidomimetics.
[0088] Aspects of the present invention provide, in part, a
Clostridial toxin di-chain loop protease cleavage site. As used
herein, the term "Clostridial toxin di-chain loop protease cleavage
site" means means a P.sub.1--P.sub.1 scissile bond located within a
Clostridial toxin di-chain loop region, together with adjacent or
non-adjacent recognition elements, or both, sufficient for
detectable proteolysis at the scissile bond by a Clostridial toxin
di-chain loop protease under conditions suitable for Clostridial
toxin di-chain loop protease activity. A Clostridial toxin di-chain
loop region includes, without limitation, a BoNT/A di-chain loop
protease cleavage site, a BoNT/B di-chain loop protease cleavage
site, a BoNT/C1 di-chain loop protease cleavage site, a BoNT/D
di-chain loop protease cleavage site, a BoNT/E di-chain loop
protease cleavage site, a BoNT/F di-chain loop protease cleavage
site, a BoNT/G di-chain loop protease cleavage site, a TeNT
di-chain loop protease cleavage site, a BaNT di-chain loop protease
cleavage site, and a BuNT di-chain loop protease cleavage site.
Non-limiting examples of a BoNT/A di-chain loop protease cleavage
site include the S441-L442 scissile bond and the K448-A449 scissile
bond. Non-limiting examples of a BoNT/B di-chain loop protease
cleavage site include the K441-A442 scissile bond and the G444-1445
scissile bond. Non-limiting examples of a BoNT/C1 di-chain loop
protease cleavage site include the S445-L446 scissile bond and the
K449-T450 scissile bond. Non-limiting examples of a BoNT/D di-chain
loop protease cleavage site include the K442-N443 scissile bond and
the R445-D446 scissile bond. Non-limiting examples of a BoNT/E
di-chain loop protease cleavage site include the K419-G420 scissile
bond, the R422-K423 scissile bond, and the K423-S424 scissile bond.
Non-limiting examples of a BoNT/F di-chain loop protease cleavage
site include the K436-G437 scissile bond and the K439-A440 scissile
bond. Non-limiting examples of a BoNT/G di-chain loop protease
cleavage site include the T444-G445 scissile bond, the K446-S447
scissile bond, and the E448-Q449 scissile bond. A non-limiting
example of a TeNT di-chain loop protease cleavage site is the
A457-S458 scissile bond. A non-limiting example of a BaNT di-chain
loop protease cleavage site is the K431-N432 scissile bond. A
non-limiting example of a BuNT di-chain loop protease cleavage site
is the R422-K423 scissile bond.
[0089] Thus, in an embodiment, a modified Clostridial toxin
comprises a BoNT/A di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BoNT/A
di-chain loop region including a BoNT/A di-chain loop protease
cleavage site. In another aspect of this embodiment, a modified
Clostridial toxin comprises a BoNT/A di-chain loop region including
a BoNT/A di-chain loop protease cleavage site comprising the
S441-L442 scissile bond. In another aspect of this embodiment, a
modified Clostridial toxin comprises a BoNT/A di-chain loop region
including a BoNT/A di-chain loop protease cleavage site comprising
the K448-A449 scissile bond. In yet another aspect of this
embodiment, a modified Clostridial toxin comprises the BoNT/A
di-chain loop region of SEQ ID NO: 11.
[0090] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BoNT/A di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BoNT/A di-chain
loop region variant, such as, e.g., a BoNT/A di-chain loop region
isoform, or a BoNT/A di-chain loop region subtype. In another
aspect of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BoNT/A di-chain loop region variant of SEQ ID
NO: 11, such as, e.g., a BoNT/A di-chain loop region isoform of SEQ
ID NO: 11; or a BoNT/A di-chain loop region subtype of SEQ ID NO:
11. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BoNT/A
di-chain loop region variant, such as, e.g., a conservative BoNT/A
di-chain loop region variant, a non-conservative BoNT/A di-chain
loop region variant or a BoNT/A di-chain loop region
peptidomimetic, or any combination thereof. In still another aspect
of this embodiment, a modified Clostridial toxin comprises a
non-naturally occurring BoNT/A di-chain loop region variant of SEQ
ID NO: 11, such as, e.g., a conservative BoNT/A di-chain loop
region variant of SEQ ID NO: 11, a non-conservative BoNT/A di-chain
loop region variant of SEQ ID NO: 11 or a BoNT/A di-chain loop
region peptidomimetic of SEQ ID NO: 11, or any combination
thereof.
[0091] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/A di-chain loop region having, e.g., at
least 50% amino acid identity with SEQ ID NO: 11, at least 60%
amino acid identity with the SEQ ID NO: 11, at least 70% amino acid
identity with SEQ ID NO: 11, at least 80% amino acid identity with
SEQ ID NO: 11, or at least 90% amino acid identity with SEQ ID NO:
11. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/A di-chain loop region having,
e.g., at most 50% amino acid identity with SEQ ID NO: 11, at most
60% amino acid identity with the SEQ ID NO: 11, at most 70% amino
acid identity with SEQ ID NO: 11, at most 80% amino acid identity
with SEQ ID NO: 11, or at most 90% amino acid identity with SEQ ID
NO: 11.
[0092] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/A di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid substitutions relative to SEQ ID NO: 11.
In still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/A di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid substitutions relative to SEQ ID NO: 11.
In yet other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/A di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid additions relative to SEQ ID NO: 11. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a BoNT/A di-chain loop region having, e.g., at least one,
two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid additions relative to SEQ ID NO: 11. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/A di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid deletions relative to SEQ ID NO: 11. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/A di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid deletions relative to SEQ ID NO: 11.
[0093] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/A di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 11. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/A di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 11. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a BoNT/A di-chain loop region having, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten contiguous
amino acid additions relative to SEQ ID NO: 11. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/A di-chain loop region having, e.g., at least one, two,
three, four, five, six, seven, eight, nine or ten contiguous amino
acid additions relative to SEQ ID NO: 11. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/A
di-chain loop region having, e.g., at most one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid
deletions relative to SEQ ID NO: 11. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BoNT/A
di-chain loop region having, e.g., at least one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid
deletions relative to SEQ ID NO: 11.
[0094] In another embodiment, a modified Clostridial toxin
comprises a BoNT/B di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BoNT/B
di-chain loop region including a BoNT/B di-chain loop protease
cleavage site. In another aspect of this embodiment, a modified
Clostridial toxin comprises a BoNT/B di-chain loop region including
a BoNT/B di-chain loop protease cleavage site comprising the
K441-A442 scissile bond. In another aspect of this embodiment, a
modified Clostridial toxin comprises a BoNT/B di-chain loop region
including a BoNT/B di-chain loop protease cleavage site comprising
the G444-1445 scissile bond. In yet another aspect of this
embodiment, a modified Clostridial toxin comprises the BoNT/B
di-chain loop region of SEQ ID NO: 12.
[0095] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BoNT/B di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BoNT/B di-chain
loop region variant, such as, e.g., a BoNT/B di-chain loop region
isoform, or a BoNT/B di-chain loop region subtype. In another
aspect of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BoNT/B di-chain loop region variant of SEQ ID
NO: 12, such as, e.g., a BoNT/B di-chain loop region isoform of SEQ
ID NO: 12; or a BoNT/B di-chain loop region subtype of SEQ ID NO:
12. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BoNT/B
di-chain loop region variant, such as, e.g., a conservative BoNT/B
di-chain loop region variant, a non-conservative BoNT/B di-chain
loop region variant or a BoNT/B di-chain loop region
peptidomimetic, or any combination thereof. In still another aspect
of this embodiment, a modified Clostridial toxin comprises a
non-naturally occurring BoNT/B di-chain loop region variant of SEQ
ID NO: 12, such as, e.g., a conservative BoNT/B di-chain loop
region variant of SEQ ID NO: 12, a non-conservative BoNT/B di-chain
loop region variant of SEQ ID NO: 12 or a BoNT/B di-chain loop
region peptidomimetic of SEQ ID NO: 12, or any combination
thereof.
[0096] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/B di-chain loop region having, e.g., at
least 50% amino acid identity with SEQ ID NO: 12, at least 60%
amino acid identity with the SEQ ID NO: 12, at least 70% amino acid
identity with SEQ ID NO: 12, at least 80% amino acid identity with
SEQ ID NO: 12, or at least 90% amino acid identity with SEQ ID NO:
12. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/B di-chain loop region having,
e.g., at most 50% amino acid identity with SEQ ID NO: 12, at most
60% amino acid identity with the SEQ ID NO: 12, at most 70% amino
acid identity with SEQ ID NO: 12, at most 80% amino acid identity
with SEQ ID NO: 12, or at most 90% amino acid identity with SEQ ID
NO: 12.
[0097] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/B di-chain loop region having, e.g., at most
one, two, three, four, or five non-contiguous amino acid
substitutions relative to SEQ ID NO: 12. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/B
di-chain loop region having, e.g., at least one, two, three, four,
or five non-contiguous amino acid substitutions relative to SEQ ID
NO: 12. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/B di-chain loop region having,
e.g., at most one, two, three, four, or five non-contiguous amino
acid additions relative to SEQ ID NO: 12. In yet other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/B
di-chain loop region having, e.g., at least one, two, three, four,
or five non-contiguous amino acid additions relative to SEQ ID NO:
12. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/B di-chain loop region having,
e.g., at most one, two, three, four, or five non-contiguous amino
acid deletions relative to SEQ ID NO: 12. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/B
di-chain loop region having, e.g., at least one, two, three, four,
or five non-contiguous amino acid deletions relative to SEQ ID NO:
12.
[0098] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/B di-chain loop region having, e.g., at most
one, two, three, four, or five contiguous amino acid substitutions
relative to SEQ ID NO: 12. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BoNT/B
di-chain loop region having, e.g., at least one, two, three, four,
or five contiguous amino acid substitutions relative to SEQ ID NO:
12. In yet other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/B di-chain loop region having, e.g., at most
one, two, three, four, or five contiguous amino acid additions
relative to SEQ ID NO: 12. In yet other aspects of this embodiment,
a modified Clostridial toxin comprises a BoNT/B di-chain loop
region having, e.g., at least one, two, three, four, or five
contiguous amino acid additions relative to SEQ ID NO: 12. In still
other aspects of this embodiment, a modified Clostridial toxin
comprises a BoNT/B di-chain loop region having, e.g., at most one,
two, three, four, or five contiguous amino acid deletions relative
to SEQ ID NO: 12. In still other aspects of this embodiment, a
modified Clostridial toxin comprises a BoNT/B di-chain loop region
having, e.g., at least one, two, three, four, or five contiguous
amino acid deletions relative to SEQ ID NO: 12.
[0099] In another embodiment, a modified Clostridial toxin
comprises a BoNT/C1 di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BoNT/C1
di-chain loop region including a BoNT/C1 di-chain loop protease
cleavage site. In another aspect of this embodiment, a modified
Clostridial toxin comprises a BoNT/C1 di-chain loop region
including a BoNT/C1 di-chain loop protease cleavage site comprising
the 5445-L446 scissile bond. In another aspect of this embodiment,
a modified Clostridial toxin comprises a BoNT/C1 di-chain loop
region including a BoNT/C1 di-chain loop protease cleavage site
comprising the K449-T450 scissile bond. In yet another aspect of
this embodiment, a modified Clostridial toxin comprises the BoNT/C1
di-chain loop region of SEQ ID NO: 13.
[0100] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BoNT/C1 di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BoNT/C1 di-chain
loop region variant, such as, e.g., a BoNT/C1 di-chain loop region
isoform, or a BoNT/C1 di-chain loop region subtype. In another
aspect of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BoNT/C1 di-chain loop region variant of SEQ ID
NO: 13, such as, e.g., a BoNT/C1 di-chain loop region isoform of
SEQ ID NO: 13; or a BoNT/C1 di-chain loop region subtype of SEQ ID
NO: 13. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BoNT/C1
di-chain loop region variant, such as, e.g., a conservative BoNT/C1
di-chain loop region variant, a non-conservative BoNT/C1 di-chain
loop region variant or a BoNT/C1 di-chain loop region
peptidomimetic, or any combination thereof. In still another aspect
of this embodiment, a modified Clostridial toxin comprises a
non-naturally occurring BoNT/C1 di-chain loop region variant of SEQ
ID NO: 13, such as, e.g., a conservative BoNT/C1 di-chain loop
region variant of SEQ ID NO: 13, a non-conservative BoNT/C1
di-chain loop region variant of SEQ ID NO: 13 or a BoNT/C1 di-chain
loop region peptidomimetic of SEQ ID NO: 13, or any combination
thereof.
[0101] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/C1 di-chain loop region having, e.g., at
least 50% amino acid identity with SEQ ID NO: 13, at least 60%
amino acid identity with the SEQ ID NO: 13, at least 70% amino acid
identity with SEQ ID NO: 13, at least 80% amino acid identity with
SEQ ID NO: 13, or at least 90% amino acid identity with SEQ ID NO:
13. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/C1 di-chain loop region having,
e.g., at most 50% amino acid identity with SEQ ID NO: 13, at most
60% amino acid identity with the SEQ ID NO: 13, at most 70% amino
acid identity with SEQ ID NO: 13, at most 80% amino acid identity
with SEQ ID NO: 13, or at most 90% amino acid identity with SEQ ID
NO: 13.
[0102] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/C1 di-chain loop region having, e.g., at
most one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid substitutions relative to SEQ ID NO: 13.
In still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/C1 di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid substitutions relative to SEQ ID NO: 13.
In yet other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/C1 di-chain loop region having, e.g., at
most one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid additions relative to SEQ ID NO: 13. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a BoNT/C1 di-chain loop region having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid additions relative to SEQ ID NO: 13. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/C1 di-chain loop region having, e.g., at
most one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid deletions relative to SEQ ID NO: 13. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/C1 di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid deletions relative to SEQ ID NO: 13.
[0103] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/C1 di-chain loop region having, e.g., at
most one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 13. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/C1 di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 13. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a BoNT/C1 di-chain loop region having, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten contiguous
amino acid additions relative to SEQ ID NO: 13. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/C1 di-chain loop region having, e.g., at least one, two,
three, four, five, six, seven, eight, nine or ten contiguous amino
acid additions relative to SEQ ID NO: 13. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/C1
di-chain loop region having, e.g., at most one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid
deletions relative to SEQ ID NO: 13. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BoNT/C1
di-chain loop region having, e.g., at least one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid
deletions relative to SEQ ID NO: 13.
[0104] In another embodiment, a modified Clostridial toxin
comprises a BoNT/D di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BoNT/D
di-chain loop region including a BoNT/D di-chain loop protease
cleavage site. In another aspect of this embodiment, a modified
Clostridial toxin comprises a BoNT/D di-chain loop region including
a BoNT/D di-chain loop protease cleavage site comprising the
K442-N443 scissile bond. In another aspect of this embodiment, a
modified Clostridial toxin comprises a BoNT/D di-chain loop region
including a BoNT/D di-chain loop protease cleavage site comprising
the R445-D446 scissile bond. In yet another aspect of this
embodiment, a modified Clostridial toxin comprises the BoNT/D
di-chain loop region of SEQ ID NO: 14.
[0105] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BoNT/D di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BoNT/D di-chain
loop region variant, such as, e.g., a BoNT/D di-chain loop region
isoform, or a BoNT/D di-chain loop region subtype. In another
aspect of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BoNT/D di-chain loop region variant of SEQ ID
NO: 14, such as, e.g., a BoNT/D di-chain loop region isoform of SEQ
ID NO: 14; or a BoNT/D di-chain loop region subtype of SEQ ID NO:
14. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BoNT/D
di-chain loop region variant, such as, e.g., a conservative BoNT/D
di-chain loop region variant, a non-conservative BoNT/D di-chain
loop region variant or a BoNT/D di-chain loop region
peptidomimetic, or any combination thereof. In still another aspect
of this embodiment, a modified Clostridial toxin comprises a
non-naturally occurring BoNT/D di-chain loop region variant of SEQ
ID NO: 14, such as, e.g., a conservative BoNT/D di-chain loop
region variant of SEQ ID NO: 14, a non-conservative BoNT/D di-chain
loop region variant of SEQ ID NO: 14 or a BoNT/D di-chain loop
region peptidomimetic of SEQ ID NO: 14, or any combination
thereof.
[0106] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/D di-chain loop region having, e.g., at
least 50% amino acid identity with SEQ ID NO: 14, at least 60%
amino acid identity with the SEQ ID NO: 14, at least 70% amino acid
identity with SEQ ID NO: 14, at least 80% amino acid identity with
SEQ ID NO: 14, or at least 90% amino acid identity with SEQ ID NO:
14. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/D di-chain loop region having,
e.g., at most 50% amino acid identity with SEQ ID NO: 14, at most
60% amino acid identity with the SEQ ID NO: 14, at most 70% amino
acid identity with SEQ ID NO: 14, at most 80% amino acid identity
with SEQ ID NO: 14, or at most 90% amino acid identity with SEQ ID
NO: 14.
[0107] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/D di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven non-contiguous amino
acid substitutions relative to SEQ ID NO: 14. In still other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/D di-chain loop region having, e.g., at least one, two,
three, four, five, six, or seven non-contiguous amino acid
substitutions relative to SEQ ID NO: 14. In yet other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/D
di-chain loop region having, e.g., at most one, two, three, four,
five, six, or seven non-contiguous amino acid additions relative to
SEQ ID NO: 14. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/D di-chain loop region having,
e.g., at least one, two, three, four, five, six, or seven
non-contiguous amino acid additions relative to SEQ ID NO: 14. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/D di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven non-contiguous amino
acid deletions relative to SEQ ID NO: 14. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/D
di-chain loop region having, e.g., at least one, two, three, four,
five, six, or seven non-contiguous amino acid deletions relative to
SEQ ID NO: 14.
[0108] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/D di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven contiguous amino acid
substitutions relative to SEQ ID NO: 14. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/D
di-chain loop region having, e.g., at least one, two, three, four,
five, six, or seven contiguous amino acid substitutions relative to
SEQ ID NO: 14. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/D di-chain loop region having,
e.g., at most one, two, three, four, five, six, or seven contiguous
amino acid additions relative to SEQ ID NO: 14. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/D di-chain loop region having, e.g., at least one, two,
three, four, five, six, or seven contiguous amino acid additions
relative to SEQ ID NO: 14. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BoNT/D
di-chain loop region having, e.g., at most one, two, three, four,
five, six, or seven contiguous amino acid deletions relative to SEQ
ID NO: 14. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/D di-chain loop region having,
e.g., at least one, two, three, four, five, six, or seven
contiguous amino acid deletions relative to SEQ ID NO: 14.
[0109] In another embodiment, a modified Clostridial toxin
comprises a BoNT/E di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BoNT/E
di-chain loop region including a BoNT/E di-chain loop protease
cleavage site. In another aspect of this embodiment, a modified
Clostridial toxin comprises a BoNT/E di-chain loop region including
a BoNT/E di-chain loop protease cleavage site comprising the
K419-G420 scissile bond. In another aspect of this embodiment, a
modified Clostridial toxin comprises a BoNT/E di-chain loop region
including a BoNT/E di-chain loop protease cleavage site comprising
the R422-K423 scissile bond. In another aspect of this embodiment,
a modified Clostridial toxin comprises a BoNT/E di-chain loop
region including a BoNT/E di-chain loop protease cleavage site
comprising the K423-S424 scissile bond. In yet another aspect of
this embodiment, a modified Clostridial toxin comprises the BoNT/E
di-chain loop region of SEQ ID NO: 15.
[0110] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BoNT/E di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BoNT/E di-chain
loop region variant, such as, e.g., a BoNT/E di-chain loop region
isoform, or a BoNT/E di-chain loop region subtype. In another
aspect of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BoNT/E di-chain loop region variant of SEQ ID
NO: 15, such as, e.g., a BoNT/E di-chain loop region isoform of SEQ
ID NO: 15; or a BoNT/E di-chain loop region subtype of SEQ ID NO:
15. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BoNT/E
di-chain loop region variant, such as, e.g., a conservative BoNT/E
di-chain loop region variant, a non-conservative BoNT/E di-chain
loop region variant or a BoNT/E di-chain loop region
peptidomimetic, or any combination thereof. In still another aspect
of this embodiment, a modified Clostridial toxin comprises a
non-naturally occurring BoNT/E di-chain loop region variant of SEQ
ID NO: 15, such as, e.g., a conservative BoNT/E di-chain loop
region variant of SEQ ID NO: 15, a non-conservative BoNT/E di-chain
loop region variant of SEQ ID NO: 15 or a BoNT/E di-chain loop
region peptidomimetic of SEQ ID NO: 15, or any combination
thereof.
[0111] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/E di-chain loop region having, e.g., at
least 50% amino acid identity with SEQ ID NO: 15, at least 60%
amino acid identity with the SEQ ID NO: 15, at least 70% amino acid
identity with SEQ ID NO: 15, at least 80% amino acid identity with
SEQ ID NO: 15, or at least 90% amino acid identity with SEQ ID NO:
15. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/E di-chain loop region having,
e.g., at most 50% amino acid identity with SEQ ID NO: 15, at most
60% amino acid identity with the SEQ ID NO: 15, at most 70% amino
acid identity with SEQ ID NO: 15, at most 80% amino acid identity
with SEQ ID NO: 15, or at most 90% amino acid identity with SEQ ID
NO: 15.
[0112] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/E di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven non-contiguous amino
acid substitutions relative to SEQ ID NO: 15. In still other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/E di-chain loop region having, e.g., at least one, two,
three, four, five, six, or seven non-contiguous amino acid
substitutions relative to SEQ ID NO: 15. In yet other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/E
di-chain loop region having, e.g., at most one, two, three, four,
five, six, or seven non-contiguous amino acid additions relative to
SEQ ID NO: 15. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/E di-chain loop region having,
e.g., at least one, two, three, four, five, six, or seven
non-contiguous amino acid additions relative to SEQ ID NO: 15. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/E di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven non-contiguous amino
acid deletions relative to SEQ ID NO: 15. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/E
di-chain loop region having, e.g., at least one, two, three, four,
five, six, or seven non-contiguous amino acid deletions relative to
SEQ ID NO: 15.
[0113] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/E di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven contiguous amino acid
substitutions relative to SEQ ID NO: 15. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/E
di-chain loop region having, e.g., at least one, two, three, four,
five, six, or seven contiguous amino acid substitutions relative to
SEQ ID NO: 15. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/E di-chain loop region having,
e.g., at most one, two, three, four, five, six, or seven contiguous
amino acid additions relative to SEQ ID NO: 15. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/E di-chain loop region having, e.g., at least one, two,
three, four, five, six, or seven contiguous amino acid additions
relative to SEQ ID NO: 15. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BoNT/E
di-chain loop region having, e.g., at most one, two, three, four,
five, six, or seven contiguous amino acid deletions relative to SEQ
ID NO: 15. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/E di-chain loop region having,
e.g., at least one, two, three, four, five, six, or seven
contiguous amino acid deletions relative to SEQ ID NO: 15.
[0114] In another embodiment, a modified Clostridial toxin
comprises a BoNT/F di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BoNT/F
di-chain loop region including a BoNT/F di-chain loop protease
cleavage site. In another aspect of this embodiment, a modified
Clostridial toxin comprises a BoNT/F di-chain loop region including
a BoNT/F di-chain loop protease cleavage site comprising the
K436-G437 scissile bond. In another aspect of this embodiment, a
modified Clostridial toxin comprises a BoNT/F di-chain loop region
including a BoNT/F di-chain loop protease cleavage site comprising
the K439-A440 scissile bond. In yet another aspect of this
embodiment, a modified Clostridial toxin comprises the BoNT/F
di-chain loop region of SEQ ID NO: 16.
[0115] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BoNT/F di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BoNT/F di-chain
loop region variant, such as, e.g., a BoNT/F di-chain loop region
isoform, or a BoNT/F di-chain loop region subtype. In another
aspect of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BoNT/F di-chain loop region variant of SEQ ID
NO: 16, such as, e.g., a BoNT/F di-chain loop region isoform of SEQ
ID NO: 16; or a BoNT/F di-chain loop region subtype of SEQ ID NO:
16. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BoNT/F
di-chain loop region variant, such as, e.g., a conservative BoNT/F
di-chain loop region variant, a non-conservative BoNT/F di-chain
loop region variant or a BoNT/F di-chain loop region
peptidomimetic, or any combination thereof. In still another aspect
of this embodiment, a modified Clostridial toxin comprises a
non-naturally occurring BoNT/F di-chain loop region variant of SEQ
ID NO: 16, such as, e.g., a conservative BoNT/F di-chain loop
region variant of SEQ ID NO: 16, a non-conservative BoNT/F di-chain
loop region variant of SEQ ID NO: 16 or a BoNT/F di-chain loop
region peptidomimetic of SEQ ID NO: 16, or any combination
thereof.
[0116] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/F di-chain loop region having, e.g., at
least 50% amino acid identity with SEQ ID NO: 16, at least 60%
amino acid identity with the SEQ ID NO: 16, at least 70% amino acid
identity with SEQ ID NO: 16, at least 80% amino acid identity with
SEQ ID NO: 16, or at least 90% amino acid identity with SEQ ID NO:
16. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/F di-chain loop region having,
e.g., at most 50% amino acid identity with SEQ ID NO: 16, at most
60% amino acid identity with the SEQ ID NO: 16, at most 70% amino
acid identity with SEQ ID NO: 16, at most 80% amino acid identity
with SEQ ID NO: 16, or at most 90% amino acid identity with SEQ ID
NO: 16.
[0117] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/F di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid substitutions relative to SEQ ID NO: 16.
In still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/F di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid substitutions relative to SEQ ID NO: 16.
In yet other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/F di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid additions relative to SEQ ID NO: 16. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a BoNT/F di-chain loop region having, e.g., at least one,
two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid additions relative to SEQ ID NO: 16. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/F di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid deletions relative to SEQ ID NO: 16. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/F di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid deletions relative to SEQ ID NO: 16.
[0118] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/F di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 16. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/F di-chain loop region having, e.g., at
least one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 16. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a BoNT/F di-chain loop region having, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten contiguous
amino acid additions relative to SEQ ID NO: 16. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/F di-chain loop region having, e.g., at least one, two,
three, four, five, six, seven, eight, nine or ten contiguous amino
acid additions relative to SEQ ID NO: 16. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/F
di-chain loop region having, e.g., at most one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid
deletions relative to SEQ ID NO: 16. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BoNT/F
di-chain loop region having, e.g., at least one, two, three, four,
five, six, seven, eight, nine or ten contiguous amino acid
deletions relative to SEQ ID NO: 16.
[0119] In another embodiment, a modified Clostridial toxin
comprises a BoNT/G di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BoNT/G
di-chain loop region including a BoNT/G di-chain loop protease
cleavage site. In another aspect of this embodiment, a modified
Clostridial toxin comprises a BoNT/G di-chain loop region including
a BoNT/G di-chain loop protease cleavage site comprising the
T444-G445 scissile bond. In another aspect of this embodiment, a
modified Clostridial toxin comprises a BoNT/G di-chain loop region
including a BoNT/G di-chain loop protease cleavage site comprising
the K446-5447 scissile bond. In another aspect of this embodiment,
a modified Clostridial toxin comprises a BoNT/G di-chain loop
region including a BoNT/G di-chain loop protease cleavage site
comprising the E448-Q449 scissile bond. In yet another aspect of
this embodiment, a modified Clostridial toxin comprises the BoNT/G
di-chain loop region of SEQ ID NO: 17.
[0120] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BoNT/G di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BoNT/G di-chain
loop region variant, such as, e.g., a BoNT/G di-chain loop region
isoform, or a BoNT/G di-chain loop region subtype. In another
aspect of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BoNT/G di-chain loop region variant of SEQ ID
NO: 17, such as, e.g., a BoNT/G di-chain loop region isoform of SEQ
ID NO: 17; or a BoNT/G di-chain loop region subtype of SEQ ID NO:
17. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BoNT/G
di-chain loop region variant, such as, e.g., a conservative BoNT/G
di-chain loop region variant, a non-conservative BoNT/G di-chain
loop region variant or a BoNT/G di-chain loop region
peptidomimetic, or any combination thereof. In still another aspect
of this embodiment, a modified Clostridial toxin comprises a
non-naturally occurring BoNT/G di-chain loop region variant of SEQ
ID NO: 17, such as, e.g., a conservative BoNT/G di-chain loop
region variant of SEQ ID NO: 17, a non-conservative BoNT/G di-chain
loop region variant of SEQ ID NO: 17 or a BoNT/G di-chain loop
region peptidomimetic of SEQ ID NO: 17, or any combination
thereof.
[0121] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/G di-chain loop region having, e.g., at
least 50% amino acid identity with SEQ ID NO: 17, at least 60%
amino acid identity with the SEQ ID NO: 17, at least 70% amino acid
identity with SEQ ID NO: 17, at least 80% amino acid identity with
SEQ ID NO: 17, or at least 90% amino acid identity with SEQ ID NO:
17. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/G di-chain loop region having,
e.g., at most 50% amino acid identity with SEQ ID NO: 17, at most
60% amino acid identity with the SEQ ID NO: 17, at most 70% amino
acid identity with SEQ ID NO: 17, at most 80% amino acid identity
with SEQ ID NO: 17, or at most 90% amino acid identity with SEQ ID
NO: 17.
[0122] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/G di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven non-contiguous amino
acid substitutions relative to SEQ ID NO: 17. In still other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/G di-chain loop region having, e.g., at least one, two,
three, four, five, six, or seven non-contiguous amino acid
substitutions relative to SEQ ID NO: 17. In yet other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/G
di-chain loop region having, e.g., at most one, two, three, four,
five, six, or seven non-contiguous amino acid additions relative to
SEQ ID NO: 17. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/G di-chain loop region having,
e.g., at least one, two, three, four, five, six, or seven
non-contiguous amino acid additions relative to SEQ ID NO: 17. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/G di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven non-contiguous amino
acid deletions relative to SEQ ID NO: 17. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/G
di-chain loop region having, e.g., at least one, two, three, four,
five, six, or seven non-contiguous amino acid deletions relative to
SEQ ID NO: 17.
[0123] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BoNT/G di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven contiguous amino acid
substitutions relative to SEQ ID NO: 17. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BoNT/G
di-chain loop region having, e.g., at least one, two, three, four,
five, six, or seven contiguous amino acid substitutions relative to
SEQ ID NO: 17. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/G di-chain loop region having,
e.g., at most one, two, three, four, five, six, or seven contiguous
amino acid additions relative to SEQ ID NO: 17. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a BoNT/G di-chain loop region having, e.g., at least one, two,
three, four, five, six, or seven contiguous amino acid additions
relative to SEQ ID NO: 17. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BoNT/G
di-chain loop region having, e.g., at most one, two, three, four,
five, six, or seven contiguous amino acid deletions relative to SEQ
ID NO: 17. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BoNT/G di-chain loop region having,
e.g., at least one, two, three, four, five, six, or seven
contiguous amino acid deletions relative to SEQ ID NO: 17.
[0124] In another embodiment, a modified Clostridial toxin
comprises a TeNT di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a TeNT di-chain
loop region including a TeNT di-chain loop protease cleavage site.
In another aspect of this embodiment, a modified Clostridial toxin
comprises a TeNT di-chain loop region including a TeNT di-chain
loop protease cleavage site comprising the A457-S458 scissile bond.
In yet another aspect of this embodiment, a modified Clostridial
toxin comprises the TeNT di-chain loop region of SEQ ID NO: 18.
[0125] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring TeNT di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring TeNT di-chain
loop region variant, such as, e.g., a TeNT di-chain loop region
isoform, or a TeNT di-chain loop region subtype. In another aspect
of this embodiment, a modified Clostridial toxin comprises a
naturally occurring TeNT di-chain loop region variant of SEQ ID NO:
18, such as, e.g., a TeNT di-chain loop region isoform of SEQ ID
NO: 18; or a TeNT di-chain loop region subtype of SEQ ID NO: 18. In
still another aspect of this embodiment, a modified Clostridial
toxin comprises a non-naturally occurring TeNT di-chain loop region
variant, such as, e.g., a conservative TeNT di-chain loop region
variant, a non-conservative TeNT di-chain loop region variant or a
TeNT di-chain loop region peptidomimetic, or any combination
thereof. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring TeNT di-chain
loop region variant of SEQ ID NO: 18, such as, e.g., a conservative
TeNT di-chain loop region variant of SEQ ID NO: 18, a
non-conservative TeNT di-chain loop region variant of SEQ ID NO: 18
or a TeNT di-chain loop region peptidomimetic of SEQ ID NO: 18, or
any combination thereof.
[0126] In other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at least
50% amino acid identity with SEQ ID NO: 18, at least 60% amino acid
identity with the SEQ ID NO: 18, at least 70% amino acid identity
with SEQ ID NO: 18, at least 80% amino acid identity with SEQ ID
NO: 18, or at least 90% amino acid identity with SEQ ID NO: 18. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at most
50% amino acid identity with SEQ ID NO: 18, at most 60% amino acid
identity with the SEQ ID NO: 18, at most 70% amino acid identity
with SEQ ID NO: 18, at most 80% amino acid identity with SEQ ID NO:
18, or at most 90% amino acid identity with SEQ ID NO: 18.
[0127] In other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid substitutions relative to SEQ ID NO: 18.
In still other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid substitutions relative to SEQ ID NO: 18.
In yet other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid additions relative to SEQ ID NO: 18. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a TeNT di-chain loop region having, e.g., at least one,
two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid additions relative to SEQ ID NO: 18. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid deletions relative to SEQ ID NO: 18. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or ten
non-contiguous amino acid deletions relative to SEQ ID NO: 18.
[0128] In other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at most
one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 18. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a TeNT di-chain loop region having, e.g., at least
one, two, three, four, five, six, seven, eight, nine or ten
contiguous amino acid substitutions relative to SEQ ID NO: 18. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a TeNT di-chain loop region having, e.g., at most one,
two, three, four, five, six, seven, eight, nine or ten contiguous
amino acid additions relative to SEQ ID NO: 18. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a TeNT di-chain loop region having, e.g., at least one, two, three,
four, five, six, seven, eight, nine or ten contiguous amino acid
additions relative to SEQ ID NO: 18. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a TeNT di-chain
loop region having, e.g., at most one, two, three, four, five, six,
seven, eight, nine or ten contiguous amino acid deletions relative
to SEQ ID NO: 18. In still other aspects of this embodiment, a
modified Clostridial toxin comprises a TeNT di-chain loop region
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine or ten contiguous amino acid deletions relative to SEQ
ID NO: 18.
[0129] In another embodiment, a modified Clostridial toxin
comprises a BaNT di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BaNT di-chain
loop region including a BaNT di-chain loop protease cleavage site.
In another aspect of this embodiment, a modified Clostridial toxin
comprises a BaNT di-chain loop region including a BaNT di-chain
loop protease cleavage site comprising the K431-N432 scissile bond.
In yet another aspect of this embodiment, a modified Clostridial
toxin comprises the BaNT di-chain loop region of SEQ ID NO: 19.
[0130] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BaNT di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BaNT di-chain
loop region variant, such as, e.g., a BaNT di-chain loop region
isoform, or a BaNT di-chain loop region subtype. In another aspect
of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BaNT di-chain loop region variant of SEQ ID NO:
19, such as, e.g., a BaNT di-chain loop region isoform of SEQ ID
NO: 19; or a BaNT di-chain loop region subtype of SEQ ID NO: 19. In
still another aspect of this embodiment, a modified Clostridial
toxin comprises a non-naturally occurring BaNT di-chain loop region
variant, such as, e.g., a conservative BaNT di-chain loop region
variant, a non-conservative BaNT di-chain loop region variant or a
BaNT di-chain loop region peptidomimetic, or any combination
thereof. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BaNT di-chain
loop region variant of SEQ ID NO: 19, such as, e.g., a conservative
BaNT di-chain loop region variant of SEQ ID NO: 19, a
non-conservative BaNT di-chain loop region variant of SEQ ID NO: 19
or a BaNT di-chain loop region peptidomimetic of SEQ ID NO: 19, or
any combination thereof.
[0131] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BaNT di-chain loop region having, e.g., at least
50% amino acid identity with SEQ ID NO: 19, at least 60% amino acid
identity with the SEQ ID NO: 19, at least 70% amino acid identity
with SEQ ID NO: 19, at least 80% amino acid identity with SEQ ID
NO: 19, or at least 90% amino acid identity with SEQ ID NO: 19. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BaNT di-chain loop region having, e.g., at most
50% amino acid identity with SEQ ID NO: 19, at most 60% amino acid
identity with the SEQ ID NO: 19, at most 70% amino acid identity
with SEQ ID NO: 19, at most 80% amino acid identity with SEQ ID NO:
19, or at most 90% amino acid identity with SEQ ID NO: 19.
[0132] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BaNT di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven non-contiguous amino
acid substitutions relative to SEQ ID NO: 19. In still other
aspects of this embodiment, a modified Clostridial toxin comprises
a BaNT di-chain loop region having, e.g., at least one, two, three,
four, five, six, or seven non-contiguous amino acid substitutions
relative to SEQ ID NO: 19. In yet other aspects of this embodiment,
a modified Clostridial toxin comprises a BaNT di-chain loop region
having, e.g., at most one, two, three, four, five, six, or seven
non-contiguous amino acid additions relative to SEQ ID NO: 19. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a BaNT di-chain loop region having, e.g., at least one,
two, three, four, five, six, or seven non-contiguous amino acid
additions relative to SEQ ID NO: 19. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BaNT di-chain
loop region having, e.g., at most one, two, three, four, five, six,
or seven non-contiguous amino acid deletions relative to SEQ ID NO:
19. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BaNT di-chain loop region having,
e.g., at least one, two, three, four, five, six, or seven
non-contiguous amino acid deletions relative to SEQ ID NO: 19.
[0133] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BaNT di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven contiguous amino acid
substitutions relative to SEQ ID NO: 19. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BaNT
di-chain loop region having, e.g., at least one, two, three, four,
five, six, or seven contiguous amino acid substitutions relative to
SEQ ID NO: 19. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BaNT di-chain loop region having,
e.g., at most one, two, three, four, five, six, or seven contiguous
amino acid additions relative to SEQ ID NO: 19. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a BaNT di-chain loop region having, e.g., at least one, two, three,
four, five, six, or seven contiguous amino acid additions relative
to SEQ ID NO: 19. In still other aspects of this embodiment, a
modified Clostridial toxin comprises a BaNT di-chain loop region
having, e.g., at most one, two, three, four, five, six, or seven
contiguous amino acid deletions relative to SEQ ID NO: 19. In still
other aspects of this embodiment, a modified Clostridial toxin
comprises a BaNT di-chain loop region having, e.g., at least one,
two, three, four, five, six, or seven contiguous amino acid
deletions relative to SEQ ID NO: 19.
[0134] In another embodiment, a modified Clostridial toxin
comprises a BuNT di-chain loop region. In an aspect of this
embodiment, a modified Clostridial toxin comprises a BuNT di-chain
loop region including a BuNT di-chain loop protease cleavage site.
In another aspect of this embodiment, a modified Clostridial toxin
comprises a BuNT di-chain loop region including a BuNT di-chain
loop protease cleavage site comprising the K431-N432 scissile bond.
In yet another aspect of this embodiment, a modified Clostridial
toxin comprises the BuNT di-chain loop region of SEQ ID NO: 20.
[0135] In another aspect of this embodiment, a modified Clostridial
toxin comprises a naturally occurring BuNT di-chain loop region
variant. In another aspect of this embodiment, a modified
Clostridial toxin comprises a naturally occurring BuNT di-chain
loop region variant, such as, e.g., a BuNT di-chain loop region
isoform, or a BuNT di-chain loop region subtype. In another aspect
of this embodiment, a modified Clostridial toxin comprises a
naturally occurring BuNT di-chain loop region variant of SEQ ID NO:
20, such as, e.g., a BuNT di-chain loop region isoform of SEQ ID
NO: 20; or a BuNT di-chain loop region subtype of SEQ ID NO: 20. In
still another aspect of this embodiment, a modified Clostridial
toxin comprises a non-naturally occurring BuNT di-chain loop region
variant, such as, e.g., a conservative BuNT di-chain loop region
variant, a non-conservative BuNT di-chain loop region variant or a
BuNT di-chain loop region peptidomimetic, or any combination
thereof. In still another aspect of this embodiment, a modified
Clostridial toxin comprises a non-naturally occurring BuNT di-chain
loop region variant of SEQ ID NO: 20, such as, e.g., a conservative
BuNT di-chain loop region variant of SEQ ID NO: 20, a
non-conservative BuNT di-chain loop region variant of SEQ ID NO: 20
or a BuNT di-chain loop region peptidomimetic of SEQ ID NO: 20, or
any combination thereof.
[0136] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BuNT di-chain loop region having, e.g., at least
50% amino acid identity with SEQ ID NO: 20, at least 60% amino acid
identity with the SEQ ID NO: 20, at least 70% amino acid identity
with SEQ ID NO: 20, at least 80% amino acid identity with SEQ ID
NO: 20, or at least 90% amino acid identity with SEQ ID NO: 20. In
still other aspects of this embodiment, a modified Clostridial
toxin comprises a BuNT di-chain loop region having, e.g., at most
50% amino acid identity with SEQ ID NO: 20, at most 60% amino acid
identity with the SEQ ID NO: 20, at most 70% amino acid identity
with SEQ ID NO: 20, at most 80% amino acid identity with SEQ ID NO:
20, or at most 90% amino acid identity with SEQ ID NO: 20.
[0137] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BuNT di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven non-contiguous amino
acid substitutions relative to SEQ ID NO: 20. In still other
aspects of this embodiment, a modified Clostridial toxin comprises
a BuNT di-chain loop region having, e.g., at least one, two, three,
four, five, six, or seven non-contiguous amino acid substitutions
relative to SEQ ID NO: 20. In yet other aspects of this embodiment,
a modified Clostridial toxin comprises a BuNT di-chain loop region
having, e.g., at most one, two, three, four, five, six, or seven
non-contiguous amino acid additions relative to SEQ ID NO: 20. In
yet other aspects of this embodiment, a modified Clostridial toxin
comprises a BuNT di-chain loop region having, e.g., at least one,
two, three, four, five, six, or seven non-contiguous amino acid
additions relative to SEQ ID NO: 20. In still other aspects of this
embodiment, a modified Clostridial toxin comprises a BuNT di-chain
loop region having, e.g., at most one, two, three, four, five, six,
or seven non-contiguous amino acid deletions relative to SEQ ID NO:
20. In still other aspects of this embodiment, a modified
Clostridial toxin comprises a BuNT di-chain loop region having,
e.g., at least one, two, three, four, five, six, or seven
non-contiguous amino acid deletions relative to SEQ ID NO: 20.
[0138] In other aspects of this embodiment, a modified Clostridial
toxin comprises a BuNT di-chain loop region having, e.g., at most
one, two, three, four, five, six, or seven contiguous amino acid
substitutions relative to SEQ ID NO: 20. In still other aspects of
this embodiment, a modified Clostridial toxin comprises a BuNT
di-chain loop region having, e.g., at least one, two, three, four,
five, six, or seven contiguous amino acid substitutions relative to
SEQ ID NO: 20. In yet other aspects of this embodiment, a modified
Clostridial toxin comprises a BuNT di-chain loop region having,
e.g., at most one, two, three, four, five, six, or seven contiguous
amino acid additions relative to SEQ ID NO: 20. In yet other
aspects of this embodiment, a modified Clostridial toxin comprises
a BuNT di-chain loop region having, e.g., at least one, two, three,
four, five, six, or seven contiguous amino acid additions relative
to SEQ ID NO: 20. In still other aspects of this embodiment, a
modified Clostridial toxin comprises a BuNT di-chain loop region
having, e.g., at most one, two, three, four, five, six, or seven
contiguous amino acid deletions relative to SEQ ID NO: 20. In still
other aspects of this embodiment, a modified Clostridial toxin
comprises a BuNT di-chain loop region having, e.g., at least one,
two, three, four, five, six, or seven contiguous amino acid
deletions relative to SEQ ID NO: 20.
[0139] The di-chain loop region of the Clostridial toxin to be
modified can be modified to include an exogenous Clostridial toxin
di-chain loop region in addition to the naturally-occurring
di-chain loop region (Table 3). In this type of modification, both
di-chain loop regions are operably-linked in-frame to the modified
Clostridial toxin as a fusion protein and both sites can be cleaved
by their respective proteases. In such a modification, the cysteine
residues from the exogenous di-chain loop region should not be
included because the additional cysteine residues could interfere
with the proper formation of the disulfide bridge necessary to for
the loop structure. As a non-limiting example, a modified BoNT/E
can comprise a di-chain loop containing both the
naturally-occurring di-chain loop region and a BoNT/A di-chain loop
region (e.g., SEQ ID NO: 11 minus the cysteine residues at position
1 and position 25) that can be cleaved by a BoNT/A di-chain loop
protease found in C. botulinum serotype A.
TABLE-US-00003 TABLE 3 Examples of Modified Clostridial Toxins
Di-Chain Enzymatic Domain Loop Region.sup.1 Translocation Domain
Binding Domain BoNT/B, BoNT/C1, BoNT/A BoNT/B, BoNT/C1, BoNT/D,
BoNT/B, BoNT/C1, BoNT/D, BoNT/D, BoNT/E, BoNT/E, BoNT/F, BoNT/G,
BoNT/E, BoNT/F, BoNT/G, BoNT/F, BoNT/G, TeNT, TeNT, BaNT, or BuNT
TeNT, BaNT, BuNT, or BaNT, or BuNT targeting moiety.sup.2 BoNT/A,
BoNT/C1, BoNT/B BoNT/A, BoNT/C1 , BoNT/D, BoNT/A, BoNT/C1, BoNT/D,
BoNT/D, BoNT/E, BoNT/E, BoNT/F, BoNT/G, BoNT/E, BoNT/F, BoNT/G,
BoNT/F, BoNT/G, TeNT, TeNT, BaNT, or BuNT TeNT, BaNT, BuNT, or re-
BaNT, or BuNT targeting moeity BoNT/A, BoNT/B, BoNT/C1 BoNT/A,
BoNT/B, BoNT/D, BoNT/A, BoNT/B, BoNT/D, BoNT/D, BoNT/E, BoNT/E,
BoNT/F, BoNT/G, BoNT/E, BoNT/F, BoNT/G, BoNT/F, BoNT/G, TeNT, TeNT,
BaNT, or BuNT TeNT, BaNT, BuNT, or re- BaNT, or BuNT targeting
moeity BoNT/A, BoNT/B, BoNT/D BoNT/A, BoNT/B, BoNT/C1, BoNT/A,
BoNT/B, BoNT/C1, BoNT/C1, BoNT/E, BoNT/E, BoNT/F, BoNT/G, BoNT/E,
BoNT/F, BoNT/G, BoNT/F, BoNT/G, TeNT, TeNT, BaNT, or BuNT TeNT,
BaNT, BuNT, or re- BaNT, or BuNT targeting moeity BoNT/A, BoNT/B,
BoNT/E BoNT/A, BoNT/B, BoNT/C1, BoNT/A, BoNT/B, BoNT/C1, BoNT/C1,
BoNT/D, BoNT/D, BoNT/F, BoNT/G, BoNT/D, BoNT/F, BoNT/G, BoNT/F,
BoNT/G, TeNT, TeNT, BaNT, or BuNT TeNT, BaNT, BuNT, or re- BaNT, or
BuNT targeting moeity BoNT/A, BoNT/B, BoNT/F BoNT/A, BoNT/B,
BoNT/C1, BoNT/A, BoNT/B, BoNT/C1, BoNT/C1, BoNT/D, BoNT/D, BoNT/E,
BoNT/G, BoNT/D, BoNT/E, BoNT/G, BoNT/E, BoNT/G, TeNT, TeNT, BaNT,
or BuNT TeNT, BaNT, BuNT, or re- BaNT, or BuNT targeting moeity
BoNT/A, BoNT/B, BoNT/G BoNT/A, BoNT/B, BoNT/C1, BoNT/A, BoNT/B,
BoNT/C1, BoNT/C1, BoNT/D, BoNT/D, BoNT/E, BoNT/F, BoNT/D, BoNT/E,
BoNT/F, BoNT/E, BoNT/F, TeNT, TeNT, BaNT, or BuNT TeNT, BaNT, BuNT,
or re- BaNT, or BuNT targeting moeity BoNT/A, BoNT/B, TeNT BoNT/A,
BoNT/B, BoNT/C1, BoNT/A, BoNT/B, BoNT/C1, BoNT/C1, BoNT/D, BoNT/D,
BoNT/E, BoNT/F, BoNT/D, BoNT/E, BoNT/F, BoNT/E, BoNT/F, BoNT/G,
BaNT, or BuNT BoNT/G, BaNT, BuNT, or re- BoNT/G, BaNT, or BuNT
targeting moeity BoNT/A, BoNT/B, BaNT BoNT/A, BoNT/B, BoNT/C1,
BoNT/A, BoNT/B, BoNT/C1, BoNT/C1, BoNT/D, BoNT/D, BoNT/E, BoNT/F,
BoNT/D, BoNT/E, BoNT/F, BoNT/E, BoNT/F, BoNT/G, TeNT, or BuNT
BoNT/G, TeNT, BuNT, or re- BoNT/G, TeNT, or BuNT targeting moeity
BoNT/A, BoNT/B, BuNT BoNT/A, BoNT/B, BoNT/C1, BoNT/A, BoNT/B,
BoNT/C1, BoNT/C1, BoNT/D, BoNT/D, BoNT/E, BoNT/F, BoNT/D, BoNT/E,
BoNT/F, BoNT/E, BoNT/F, BoNT/G, TeNT, or BaNT BoNT/G, TeNT, BaNT,
or re- BoNT/G, TeNT, or BaNT targeting moeity .sup.1Included in
this category is the replacement of the endogenous Clostridial
toxin di-chain loop with the indicated exogenous Clostridial toxin
di-chain loop; replacement of the endogenous Clostridial toxin
di-chain loop protease cleavage site with the indicated exogenous
Clostridial toxin di-chain loop protease cleavage site; the
addition of an exogenous Clostridial toxin di-chain loop from the
indicated Clostridial toxin within the endogenous Clostridial toxin
di-chain loop; and the addition of an exogenous Clostridial toxin
di-chain loop protease cleavage site from the indicated Clostridial
toxin within the endogenous Clostridial toxin di-chain loop.
.sup.2Targeting moeities suitable as binding domains disclosed in
the present specification are described in Steward, supra,
International Patent Publication No. 2006/008956; Steward, supra,
U.S. patent application No. 11/776,043; Steward, supra,
International Patent Publication No. 2006/009831; Steward, supra,
U.S. Patent Publication No. 2006/0211619; Steward, supra, U.S.
patent application No. 11/776,052; Foster, supra, U.S. Pat. No.
5,989,545; Shone, supra, U.S. Pat. No. 6,461,617; Quinn, supra,
U.S. Pat. No. 6,632,440; Steward, supra, U.S. Pat. No. 6,843,998;
Donovan, supra, U.S. Pat. No. U.S. 7,138,127; Foster, supra, U.S.
Patent Publication 2003/0180289; Dolly, supra, U.S. Pat. No.
7,132,259; Foster, supra, International Patent Publication WO
2005/023309; Steward, supra, U.S. patent application No.
11/376,696; Foster, supra, International Patent Publication WO
2006/059093; Foster, supra, International Patent Publication WO
2006/059105; and Steward, supra, U.S. patent application No.
11/776,075.
[0140] The di-chain loop region of the Clostridial toxin to be
modified can be modified to include an exogenous Clostridial toxin
di-chain loop protease cleavage site in addition to the
naturally-occurring di-chain loop protease cleavage site (Table 3).
In this type of modification, both cleavage sites are
operably-linked in-frame to a modified Clostridial toxin as a
fusion protein and both sites can be cleaved by their respective
proteases. As a non-limiting example, a modified BoNT/E can
comprise a di-chain loop containing both the naturally-occurring
di-chain loop protease cleavage site and a BoNT/A di-chain loop
protease cleavage site that can be cleaved by a BoNT/A di-chain
loop protease found in C. botulinum serotype A.
[0141] The di-chain loop region can also be modified to replace the
naturally-occurring di-chain loop region with an exogenous
Clostridial toxin di-chain loop region (Table 3). Such a
Clostridial toxin di-chain loop region is operably-linked in-frame
to a modified Clostridial toxin as a fusion protein. As a
non-limiting example, a BoNT/E di-chain loop region (e.g., SEQ ID
NO: 15) can be replaced by a BoNT/A di-chain loop region (e.g., SEQ
ID NO: 11) that can be cleaved by a BoNT/A di-chain loop protease
found in C. botulinum serotype A.
[0142] The di-chain loop region can also be modified to replace a
naturally-occurring di-chain loop protease cleavage site with an
exogenous Clostridial toxin di-chain loop protease cleavage site
(Table 3). Such a Clostridial toxin di-chain loop protease cleavage
site is operably-linked in-frame to a modified Clostridial toxin as
a fusion protein. As a non-limiting example, the R422-K423 scissile
bond of a BoNT/E di-chain loop region can be replaced by a
K448-A449 scissile bond from a BoNT/A di-chain loop region that can
be cleaved by a BoNT/A di-chain loop protease found in C. botulinum
serotype A.
[0143] The naturally-occurring di-chain loop protease cleavage site
can be made inoperable by altering at least the one of the amino
acids flanking the peptide bond cleaved by the naturally-occurring
protease, i.e., either P.sub.1, P.sub.1' or both P.sub.1 and
P.sub.1'. More extensive alterations can be made, with the proviso
that the two cysteine residues of the di-chain loop region remain
intact and formation of the disulfide bridge can still be achieved.
Non-limiting examples of an amino acid alteration include deletion
of an amino acid or replacement of the original amino acid with a
different amino acid. These alterations can be made using standard
mutagenesis procedures known to a person skilled in the art. In
addition, non-limiting examples of mutagensis procedures, as well
as well-characterized reagents, conditions and protocols are
readily available from commercial vendors that include, without
limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BD
Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc,
Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; and Stratagene,
La Jolla, Calif. These protocols are routine procedures within the
scope of one skilled in the art and from the teaching herein.
[0144] Thus, in one embodiment, a naturally-occurring di-chain loop
protease cleavage site is made inoperable by altering at least one
of the amino acids flanking the peptide bond cleaved by a
naturally-occurring protease. In aspects of this embodiment, the
P.sub.1 amino acid of the di-chain loop protease cleavage site is
altered or the P.sub.1' amino acid of the di-chain loop protease
cleavage site is altered. In other aspects of this embodiment,
either K448 or A449 of BoNT/A is altered; either S441 or L442 of
BoNT/A is altered; either K441 or A442 of BoNT/B is altered; either
G444 or 1445 of BoNT/B is altered; either K449 or T450 of BoNT/C1
is altered; either S445 or L446 of BoNT/C1 is altered; either R445
or D446 of BoNT/D is altered; either K442 or N443 of BoNT/D is
altered; either R422 or K423 of BoNT/E is altered; either K419 or
G420 of BoNT/E is altered; either K423 or S424 of BoNT/E is
altered; either K439 or A440 of BoNT/F is altered; either K436 or
G437 of BoNT/F is altered; either K446 or S447 of BoNT/G is
altered; either T444 or G445 of BoNT/G is altered; either E448 or
Q449 of BoNT/G is altered; or either A457 or S458 of TeNT is
altered.
[0145] In another embodiment, a naturally-occurring di-chain loop
protease cleavage site is made inoperable by altering the two amino
acids flanking the peptide bond cleaved by a naturally-occurring
protease, i.e., P.sub.1 and P.sub.1'. In other aspects of this
embodiment, both K448 and A449 of BoNT/A are altered; both S441 and
L442 of BoNT/A are altered; both K441 and A442 of BoNT/B are
altered; both G444 and I445 of BoNT/B are altered; both K449 and
T450 of BoNT/C1 are altered; both S445 and L446 of BoNT/C1 are
altered; both R445 and D446 of BoNT/D are altered; both K442 and
N443 of BoNT/D are altered; both R422 and K423 of BoNT/E are
altered; both K419 and G420 of BoNT/E are altered; both K423 and
S424 of BoNT/E are altered; both K439 and A440 of BoNT/F are
altered; both K436 and G437 of BoNT/F are altered; both K446 and
S447 of BoNT/G are altered; both T444 and G445 of BoNT/G are
altered; both E448 and Q449 of BoNT/G are altered; or both A457 and
S458 of TeNT are altered.
[0146] In other aspects of this embodiment, a naturally-occurring
di-chain loop protease cleavage site is made inoperable by
altering, e.g., at least two amino acids within the di-chain loop
region; at least three amino acids within the di-chain loop region;
at least four amino acids within the di-chain loop region; at least
five amino acids within the di-chain loop region; at least six
amino acids within the di-chain loop region; at least seven amino
acids within the di-chain loop region; at least eight amino acids
within the di-chain loop region; at least nine amino acids within
the di-chain loop region; at least ten amino acids within the
di-chain loop region; or at least 15 amino acids within the
di-chain loop region. In still other aspects of this embodiment, a
naturally-occurring di-chain loop protease cleavage site is made
inoperable by altering one of the amino acids flanking the peptide
bond cleaved by a naturally-occurring protease and, e.g., at least
one more amino acid within the di-chain loop region; at least two
more amino acids within the di-chain loop region; at least three
more amino acids within the di-chain loop region; at least four
more amino acids within the di-chain loop region; at least five
more amino acids within the di-chain loop region; at least six more
amino acids within the di-chain loop region; at least seven more
amino acids within the di-chain loop region; at least eight more
amino acids within the di-chain loop region; at least nine more
amino acids within the di-chain loop region; at least ten more
amino acids within the di-chain loop region; at least 15 more amino
acids within the di-chain loop region. In yet other aspects of this
embodiment, a naturally-occurring di-chain loop protease cleavage
site is made inoperable by altering the two amino acids flanking
the peptide bond cleaved by a naturally-occurring protease and,
e.g., at least one more amino acid within the di-chain loop region;
at least two more amino acids within the di-chain loop region; at
least three more amino acids within the di-chain loop region; at
least four more amino acids within the di-chain loop region; at
least five more amino acids within the di-chain loop region; at
least six more amino acids within the di-chain loop region; at
least seven more amino acids within the di-chain loop region; at
least eight more amino acids within the di-chain loop region; at
least nine more amino acids within the di-chain loop region; at
least ten more amino acids within the di-chain loop region; at
least 15 more amino acids within the di-chain loop region.
[0147] In other aspects of this embodiment, a naturally-occurring
di-chain loop protease cleavage site is made inoperable by
altering, e.g., at most two amino acids within the di-chain loop
region; at most three amino acids within the di-chain loop region;
at most four amino acids within the di-chain loop region; at most
five amino acids within the di-chain loop region; at most six amino
acids within the di-chain loop region; at most seven amino acids
within the di-chain loop region; at most eight amino acids within
the di-chain loop region; at most nine amino acids within the
di-chain loop region; at most ten amino acids within the di-chain
loop region; or at most 15 amino acids within the di-chain loop
region. In still other aspects of this embodiment, a
naturally-occurring di-chain loop protease cleavage site is made
inoperable by altering one of the amino acids flanking the peptide
bond cleaved by a naturally-occurring protease and, e.g., at most
one more amino acid within the di-chain loop region; at most two
more amino acids within the di-chain loop region; at most three
more amino acids within the di-chain loop region; at most four more
amino acids within the di-chain loop region; at most five more
amino acids within the di-chain loop region; at most six more amino
acids within the di-chain loop region; at most seven more amino
acids within the di-chain loop region; at most eight more amino
acids within the di-chain loop region; at most nine more amino
acids within the di-chain loop region; at most ten more amino acids
within the di-chain loop region; at most 15 more amino acids within
the di-chain loop region. In yet other aspects of this embodiment,
a naturally-occurring di-chain loop protease cleavage site is made
inoperable by altering the two amino acids flanking the peptide
bond cleaved by a naturally-occurring protease and, e.g., at most
one more amino acid within the di-chain loop region; at most two
more amino acids within the di-chain loop region; at most three
more amino acids within the di-chain loop region; at most four more
amino acids within the di-chain loop region; at most five more
amino acids within the di-chain loop region; at most six more amino
acids within the di-chain loop region; at most seven more amino
acids within the di-chain loop region; at most eight more amino
acids within the di-chain loop region; at most nine more amino
acids within the di-chain loop region; at most ten more amino acids
within the di-chain loop region; at most 15 more amino acids within
the di-chain loop region.
[0148] It is envisioned that the di-chain loop region of a
Clostridial toxin can be modified to include any of the other
Clostridial toxin di-chain loop regions. In aspects of this
embodiment, a Clostridial toxin di-chain loop region can be
modified to comprise, e.g., a BoNT/A di-chain loop region, a BoNT/B
di-chain loop region, a BoNT/C1 di-chain loop region, a BoNT/D
di-chain loop region, a BoNT/E di-chain loop region, a BoNT/F
di-chain loop region, a BoNT/G di-chain loop region, a TeNT
di-chain loop region, a BaNT di-chain loop region or a BuNT
di-chain loop region. In other aspects of this embodiment, an
exogenous Clostridial toxin di-chain loop region, in addition to
the naturally-occurring protease cleavage site, can be modified to
comprise, e.g., a BoNT/A di-chain loop region, a BoNT/B di-chain
loop region, a BoNT/C1 di-chain loop region, a BoNT/D di-chain loop
region, a BoNT/E di-chain loop region, a BoNT/F di-chain loop
region, a BoNT/G di-chain loop region, a TeNT di-chain loop region,
a BaNT di-chain loop region or a BuNT di-chain loop region.
[0149] In still other aspects of this embodiment, a di-chain loop
of a Clostridial toxin can be modified to replace a
naturally-occurring protease cleavage site with, e.g., a BoNT/A
substrate cleavage site, a BoNT/B substrate cleavage site, a
BoNT/C1 substrate cleavage site, a BoNT/D substrate cleavage site,
a BoNT/E substrate cleavage site, a BoNT/F substrate cleavage site,
a BoNT/G substrate cleavage site, a TeNT substrate cleavage site, a
BaNT substrate cleavage site or a BuNT substrate cleavage site.
[0150] The location of the Clostridial toxin substrate cleavage
site can be anywhere in the Clostridial toxin, with the proviso
that cleavage of the site must occur between the two cysteine
residues that form the single disulfide bridge of toxin. Thus, in
aspects of this embodiment, location of a Clostridial toxin
substrate cleavage site can be, e.g., anywhere in the BoNT/A of SEQ
ID NO: 1, with the proviso that cleavage occurs between cysteine
430 and cysteine 454; anywhere in the BoNT/B of SEQ ID NO: 2, with
the proviso that cleavage occurs between cysteine 437 and cysteine
446; anywhere in the BoNT/C1 of SEQ ID NO: 2, with the proviso that
cleavage occurs between cysteine 437 and cysteine 453; anywhere in
the BoNT/D of SEQ ID NO: 4, with the proviso that cleavage occurs
between cysteine 437 and cysteine 450; anywhere in the BoNT/E of
SEQ ID NO: 5, with the proviso that cleavage occurs between
cysteine 412 and cysteine 426; anywhere in the BoNT/F of SEQ ID NO:
6, with the proviso that cleavage occurs between cysteine 429 and
cysteine 445; anywhere in the BoNT/G of SEQ ID NO: 7, with the
proviso that cleavage occurs between cysteine 436 and cysteine 450;
or anywhere in the TeNT of SEQ ID NO: 8, with the proviso that
cleavage occurs between cysteine 439 and cysteine 467.
[0151] It is understood that a modified Clostridial toxin disclosed
in the present specification can optionally include one or more
additional components. As a non-limiting example of an optional
component, a modified Clostridial toxin can further comprise a
flexible region comprising a flexible spacer. Non-limiting examples
of a flexible spacer include, e.g., a G-spacer GGGGS (SEQ ID NO:
21) or an A-spacer EAAAK (SEQ ID NO: 22). A flexible region
comprising flexible spacers can be used to adjust the length of a
polypeptide region in order to optimize a characteristic, attribute
or property of a polypeptide. Such a flexible region is
operably-linked in-frame to the modified Clostridial toxin as a
fusion protein. As a non-limiting example, a polypeptide region
comprising one or more flexible spacers in tandem can be use to
better expose a protease cleavage site thereby facilitating
cleavage of that site by a protease. As another non-limiting
example, a polypeptide region comprising one or more flexible
spacers in tandem can be use to better present a ligand domain,
thereby facilitating the binding of that ligand domain to its
binding domain on a receptor.
[0152] Thus, in an embodiment, a modified Clostridial toxin
disclosed in the present specification can further comprise a
flexible region comprising a flexible spacer. In another
embodiment, a modified Clostridial toxin disclosed in the present
specification can further comprise flexible region comprising a
plurality of flexible spacers in tandem. In aspects of this
embodiment, a flexible region can comprise in tandem, e.g., at
least 1 G-spacer, at least 2 G-spacers, at least 3 G-spacers, at
least 4 G-spacers or at least 5 G-spacers. In other aspects of this
embodiment, a flexible region can comprise in tandem, e.g., at most
1 G-spacer, at most 2 G-spacers, at most 3 G-spacers, at most 4
G-spacers or at most 5 G-spacers. In still other aspects of this
embodiment, a flexible region can comprise in tandem, e.g., at
least 1 A-spacer, at least 2 A-spacers, at least 3 A-spacers, at
least 4 A-spacers or at least 5 A-spacers. In still other aspects
of this embodiment, a flexible region can comprise in tandem, e.g.,
at most 1 A-spacer, at most 2 A-spacers, at most 3 A-spacers, at
most 4 A-spacers or at most 5 A-spacers. In another aspect of this
embodiment, a modified Clostridial toxin can comprise a flexible
region comprising one or more copies of the same flexible spacers,
one or more copies of different flexible-spacer regions, or any
combination thereof.
[0153] As another non-limiting example of an optional component, a
modified Clostridial toxin can further comprise an epitope-binding
region. An epitope-binding region can be used in a wide variety of
procedures involving, e.g., protein purification and protein
visualization. Such an epitope-binding region is operably-linked
in-frame to a modified Clostridial toxin as a fusion protein.
Non-limiting examples of an epitope-binding region include, e.g.,
FLAG, Express.TM. (SEQ ID NO: 23), human Influenza virus
hemagluttinin (HA) (SEQ ID NO: 24), human p62.sup.c-Myc protein
(c-MYC) (SEQ ID NO: 25), Vesicular Stomatitis Virus Glycoprotein
(VSV-G) (SEQ ID NO: 26), Substance P (SEQ ID NO: 27),
glycoprotein-D precursor of Herpes simplex virus (HSV) (SEQ ID NO:
28), V5 (SEQ ID NO: 29), AU1 (SEQ ID NO: 30) and AU5 (SEQ ID NO:
31); affinity-binding, such as. e.g., polyhistidine (HIS) (SEQ ID
NO: 32), streptavidin binding peptide (strep), and biotin or a
biotinylation sequence; peptide-binding regions, such as. e.g., the
glutathione binding domain of glutathione-S-transferase, the
calmodulin binding domain of the calmodulin binding protein, and
the maltose binding domain of the maltose binding protein.
Non-limiting examples of specific protocols for selecting, making
and using an appropriate binding peptide are described in, e.g.,
Epitope Tagging, pp. 17.90-17.93 (Sambrook and Russell, eds.,
MOLECULAR CLONING A LABORATORY MANUAL, VOl. 3, 3.sup.rd ed. 2001);
ANTIBODIES: A LABORATORY MANUAL (Edward Harlow & David Lane,
eds., Cold Spring Harbor Laboratory Press, 2.sup.nd ed. 1998); and
USING ANTIBODIES: A LABORATORY MANUAL: PORTABLE PROTOCOL NO. I
(Edward Harlow & David Lane, Cold Spring Harbor Laboratory
Press, 1998). In addition, non-limiting examples of binding
peptides as well as well-characterized reagents, conditions and
protocols are readily available from commercial vendors that
include, without limitation, BD Biosciences-Clontech, Palo Alto,
Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen,
Inc, Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; and
Stratagene, La Jolla, Calif. These protocols are routine procedures
well within the scope of one skilled in the art and from the
teaching herein.
[0154] Thus, in an embodiment, a modified Clostridial toxin
disclosed in the present specification can further comprise an
epitope-binding region. In another embodiment, a modified
Clostridial toxin disclosed in the present specification can
further comprises a plurality of epitope-binding regions. In
aspects of this embodiment, a modified Clostridial toxin can
comprise, e.g., at least 1 epitope-binding region, at least 2
epitope-binding regions, at least 3 epitope-binding regions, at
least 4 epitope-binding regions or at least 5 epitope-binding
regions. In other aspects of this embodiment, a modified
Clostridial toxin can comprise, e.g., at most 1 epitope-binding
region, at most 2 epitope-binding regions, at most 3
epitope-binding regions, at most 4 epitope-binding regions or at
most 5 epitope-binding regions. In another aspect of this
embodiment, a modified Clostridial toxin can comprise one or more
copies of the same epitope-binding region, one or more copies of
different epitope-binding regions, or any combination thereof.
[0155] The location of an epitope-binding region can be in various
positions, including, without limitation, at the amino terminus of
a modified Clostridial toxin, within a modified Clostridial toxin,
or at the carboxyl terminus of a modified Clostridial toxin. Thus,
in an embodiment, an epitope-binding region is located at the
amino-terminus of a modified Clostridial toxin. In such a location,
a start methionine should be placed in front of the epitope-binding
region. In addition, it is known in the art that when adding a
polypeptide that is operationally-linked to the amino terminus of
another polypeptide comprising the start methionine that the
original methionine residue can be deleted. This is due to the fact
that the added polypeptide will contain a new start methionine and
that the original start methionine may reduce optimal expression of
the fusion protein. In aspects of this embodiment, an
epitope-binding region located at the amino-terminus of a modified
Clostridial toxin disclosed in the present specification can be,
e.g., a FLAG, Express.TM. epitope-binding region, a human Influenza
virus hemagluttinin (HA) epitope-binding region, a human
p62.sup.c-Myc protein (c-MYC) epitope-binding region, a Vesicular
Stomatitis Virus Glycoprotein (VSV-G) epitope-binding region, a
Substance P epitope-binding region, a glycoprotein-D precursor of
Herpes simplex virus (HSV) epitope-binding region, a V5
epitope-binding region, a AU1 epitope-binding region, a AU5
epitope-binding region, a polyhistidine epitope-binding region, a
streptavidin binding peptide epitope-binding region, a biotin
epitope-binding region, a biotinylation epitope-binding region, a
glutathione binding domain of glutathione-S-transferase, a
calmodulin binding domain of the calmodulin binding protein or a
maltose binding domain of the maltose binding protein.
[0156] In another embodiment, an epitope-binding region is located
at the carboxyl-terminus of a modified Clostridial toxin. In
aspects of this embodiment, an epitope-binding region located at
the carboxyl-terminus of a modified Clostridial toxin disclosed in
the present specification can be, e.g., a FLAG, Express.TM.
epitope-binding region, a human Influenza virus hemagluttinin (HA)
epitope-binding region, a human p62.sup.c-Myc protein (c-MYC)
epitope-binding region, a Vesicular Stomatitis Virus Glycoprotein
(VSV-G) epitope-binding region, a Substance P epitope-binding
region, a glycoprotein-D precursor of Herpes simplex virus (HSV)
epitope-binding region, a V5 epitope-binding region, a AU1
epitope-binding region, a AU5 epitope-binding region, a
polyhistidine epitope-binding region, a streptavidin binding
peptide epitope-binding region, a biotin epitope-binding region, a
biotinylation epitope-binding region, a glutathione binding domain
of glutathione-S-transferase, a calmodulin binding domain of the
calmodulin binding protein or a maltose binding domain of the
maltose binding protein.
[0157] Aspects of the present invention provide, in part modified
Clostridial toxins. As used herein, the term "modified Clostridial
toxin" means any naturally-occurring Clostridial toxin or
non-naturally occurring Clostridial toxin comprising at least 1)
the replacement of a naturally-occurring di-chain loop protease
cleavage site with a di-chain loop protease cleavage site from
another Clostridial toxin, 2) the addition of a Clostridial toxin
di-chain loop protease cleavage site as disclosed in the present
specification into the di-chain loop region of a
naturally-occurring Clostridial toxin, 3) the replacement of a
naturally-occurring di-chain loop region with a di-chain loop
region from another Clostridial toxin, or 4) the addition of a
Clostridial toxin di-chain loop region as disclosed in the present
specification into the di-chain loop region of a
naturally-occurring Clostridial toxin.
[0158] It is understood that all such modifications do not
substantially affect the ability of a Clostridial toxin to
intoxicate a cell. As used herein, the term "do not substantially
affect" means a Clostridial toxin can still execute the overall
cellular mechanism whereby a Clostridial toxin enters a neuron and
inhibits neurotransmitter release and encompasses the binding of a
Clostridial toxin to a low or high affinity receptor complex, the
internalization of the toxin/receptor complex, the translocation of
the Clostridial toxin light chain into the cytoplasm and the
enzymatic modification of a Clostridial toxin substrate. In aspects
of this embodiment, the modified Clostridial toxin is, e.g., at
least 10% as toxic as a naturally-occurring Clostridial toxin, at
least 20% as toxic as a naturally-occurring Clostridial toxin, at
least 30% as toxic as a naturally-occurring Clostridial toxin, at
least 40% as toxic as a naturally-occurring Clostridial toxin, at
least 50% as toxic as a naturally-occurring Clostridial toxin, at
least 60% as toxic as a naturally-occurring Clostridial toxin, at
least 70% as toxic as a naturally-occurring Clostridial toxin, at
least 80% as toxic as a naturally-occurring Clostridial toxin, at
least 90% as toxic as a naturally-occurring Clostridial toxin or at
least 95% as toxic as a naturally-occurring Clostridial toxin. In
aspects of this embodiment, the modified Clostridial toxin is,
e.g., at most 10% as toxic as a naturally-occurring Clostridial
toxin, at most 20% as toxic as a naturally-occurring Clostridial
toxin, at most 30% as toxic as a naturally-occurring Clostridial
toxin, at most 40% as toxic as a naturally-occurring Clostridial
toxin, at most 50% as toxic as a naturally-occurring Clostridial
toxin, at most 60% as toxic as a naturally-occurring Clostridial
toxin, at most 70% as toxic as a naturally-occurring Clostridial
toxin, at most 80% as toxic as a naturally-occurring Clostridial
toxin, at most 90% as toxic as a naturally-occurring Clostridial
toxin or at most 95% as toxic as a naturally-occurring Clostridial
toxin.
[0159] Aspects of the present invention provide, in part,
polynucleotide molecules. As used herein, the term "polynucleotide
molecule" is synonymous with "nucleic acid molecule" and means a
polymeric form of nucleotides, such as, e.g., ribonucleotides and
deoxyribonucleotides, of any length. It is envisioned that any and
all polynucleotide molecules that can encode a modified Clostridial
toxin disclosed in the present specification can be useful,
including, without limitation naturally-occurring and
non-naturally-occurring DNA molecules and naturally-occurring and
non-naturally-occurring RNA molecules. Non-limiting examples of
naturally-occurring and non-naturally-occurring DNA molecules
include single-stranded DNA molecules, double-stranded DNA
molecules, genomic DNA molecules, cDNA molecules, vector
constructs, such as, e.g., plasmid constructs, phagmid constructs,
bacteriophage constructs, retroviral constructs and artificial
chromosome constructs. Non-limiting examples of naturally-occurring
and non-naturally-occurring RNA molecules include single-stranded
RNA, double stranded RNA and mRNA.
[0160] Well-established molecular biology techniques that may be
necessary to make a polynucleotide molecule encoding a modified
Clostridial toxin disclosed in the present specification including,
but not limited to, procedures involving polymerase chain reaction
(PCR) amplification, restriction enzyme reactions, agarose gel
electrophoresis, nucleic acid ligation, bacterial transformation,
nucleic acid purification, nucleic acid sequencing and
recombination-based techniques are routine procedures well within
the scope of one skilled in the art and from the teaching herein.
Non-limiting examples of specific protocols necessary to make a
polynucleotide molecule encoding a modified Clostridial toxin are
described in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra,
(2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M.
Ausubel et al., eds. John Wiley & Sons, 2004). Additionally, a
variety of commercially available products useful for making a
polynucleotide molecule encoding a modified Clostridial toxin are
widely available. These protocols are routine procedures well
within the scope of one skilled in the art and from the teaching
herein.
[0161] Another aspect of the present invention provides a method of
producing a modified Clostridial toxin comprising an exogenous
Clostridial toxin di-chain loop region including a Clostridial
toxin di-chain loop protease cleavage site from a different
Clostridial toxin, such method comprising the step of expressing a
polynucleotide molecule encoding a modified Clostridial toxin in a
cell. Another aspect of the present invention provides a method of
producing a modified Clostridial toxin comprising an exogenous
Clostridial toxin di-chain loop region including a Clostridial
toxin di-chain loop protease cleavage site from a different
Clostridial toxin, such method comprising the steps of introducing
an expression construct comprising a polynucleotide molecule
encoding the modified Clostridial toxin into a cell and expressing
the expression construct in the cell.
[0162] The methods disclosed in the present specification include,
in part, a modified Clostridial toxin. It is envisioned that any
and all modified Clostridial toxins disclosed in the present
specification can be produced using the methods disclosed in the
present specification. Thus, aspects of this embodiment include
producing, without limitation, naturally occurring Clostridial
toxins, naturally occurring Clostridial toxins variants, such as,
e.g., Clostridial toxins isoforms and Clostridial toxins subtypes,
non-naturally occurring Clostridial toxins variants, such as, e.g.,
conservative Clostridial toxins variants, non-conservative
Clostridial toxins variants and Clostridial toxins fragments
thereof, or any combination thereof.
[0163] The methods disclosed in the present specification include,
in part, a polynucleotide molecule. It is envisioned that any and
all polynucleotide molecules disclosed in the present specification
can be used. Thus, aspects of this embodiment include, without
limitation, naturally-occurring and non-naturally-occurring DNA
molecules include single-stranded DNA molecules, double-stranded
DNA molecules, genomic DNA molecules, cDNA molecules, vector
constructs, such as, e.g., plasmid constructs, phagmid constructs,
bacteriophage constructs, retroviral constructs and artificial
chromosome constructs. Non-limiting examples of naturally-occurring
and non-naturally-occurring RNA molecules include single-stranded
RNA, double stranded RNA and mRNA.
[0164] The methods disclosed in the present specification include,
in part, an expression construct. An expression construct comprises
a polynucleotide molecule disclosed in the present specification
operably-linked to an expression vector useful for expressing the
polynucleotide molecule in a cell or cell-free extract. A wide
variety of expression vectors can be employed for expressing a
polynucleotide molecule encoding a modified Clostridial toxin,
including, without limitation, a viral expression vector; a
prokaryotic expression vector; eukaryotic expression vectors, such
as, e.g., a yeast expression vector, an insect expression vector
and a mammalian expression vector; and a cell-free extract
expression vector. It is further understood that expression vectors
useful to practice aspects of these methods may include those which
express a modified Clostridial toxin under control of a
constitutive, tissue-specific, cell-specific or inducible promoter
element, enhancer element or both. Non-limiting examples of
expression vectors, along with well-established reagents and
conditions for making and using an expression construct from such
expression vectors are readily available from commercial vendors
that include, without limitation, BD Biosciences-Clontech, Palo
Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.;
Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences-Novagen,
Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La
Jolla, Calif. The selection, making and use of an appropriate
expression vector are routine procedures well within the scope of
one skilled in the art and from the teachings herein.
[0165] Thus, aspects of this embodiment include, without
limitation, a viral expression vector operably-linked to a
polynucleotide molecule encoding a modified Clostridial toxin; a
prokaryotic expression vector operably-linked to a polynucleotide
molecule encoding a modified Clostridial toxin; a yeast expression
vector operably-linked to a polynucleotide molecule encoding a
modified Clostridial toxin; an insect expression vector
operably-linked to a polynucleotide molecule encoding a modified
Clostridial toxin; and a mammalian expression vector
operably-linked to a polynucleotide molecule encoding a modified
Clostridial toxin. Other aspects of this embodiment include,
without limitation, expression constructs suitable for expressing a
modified Clostridial toxin disclosed in the present specification
using a cell-free extract comprising a cell-free extract expression
vector operably linked to a polynucleotide molecule encoding a
modified Clostridial toxin.
[0166] The methods disclosed in the present specification include,
in part, a cell. It is envisioned that any and all cells can be
used. Thus, aspects of this embodiment include, without limitation,
prokaryotic cells including, without limitation, strains of
aerobic, microaerophilic, capnophilic, facultative, anaerobic,
gram-negative and gram-positive bacterial cells such as those
derived from, e.g., Escherichia coli, Bacillus subtilis, Bacillus
licheniformis, Bacteroides fragilis, Clostridia perfringens,
Clostridia difficile, Caulobacter crescentus, Lactococcus lactis,
Methylobacterium extorquens, Neisseria meningirulls, Neisseria
meningitidis, Pseudomonas fluorescens and Salmonella typhimurium;
and eukaryotic cells including, without limitation, yeast strains,
such as, e.g., those derived from Pichia pastoris, Pichia
methanolica, Pichia angusta, Schizosaccharomyces pombe,
Saccharomyces cerevisiae and Yarrowia lipolytica; insect cells and
cell lines derived from insects, such as, e.g., those derived from
Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster and
Manduca Sexta; and mammalian cells and cell lines derived from
mammalian cells, such as, e.g., those derived from mouse, rat,
hamster, porcine, bovine, equine, primate and human. Cell lines may
be obtained from the American Type Culture Collection (2004);
European Collection of Cell Cultures (2204); and the German
Collection of Microorganisms and Cell Cultures (2004). Non-limiting
examples of specific protocols for selecting, making and using an
appropriate cell line are described in e.g., INSECT CELL CULTURE
ENGINEERING (Mattheus F. A. Goosen et al. eds., Marcel Dekker,
1993); INSECT CELL CULTURES: FUNDAMENTAL AND APPLIED ASPECTS (J. M.
Vlak et al. eds., Kluwer Academic Publishers, 1996); Maureen A.
Harrison & Ian F. Rae, GENERAL TECHNIQUES OF CELL CULTURE
(Cambridge University Press, 1997); CELL AND TISSUE CULTURE:
LABORATORY PROCEDURES (Alan Doyle et al eds., John Wiley and Sons,
1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC
TECHNIQUE (Wiley-Liss, 4.sup.th ed. 2000); ANIMAL CELL CULTURE: A
PRACTICAL APPROACH (John R. W. Masters ed., Oxford University
Press, 3.sup.rd ed. 2000); MOLECULAR CLONING A LABORATORY MANUAL,
supra, (2001); BASIC CELL CULTURE: A PRACTICAL APPROACH (John M.
Davis, Oxford Press, 2.sup.nd ed. 2002); and CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, supra, (2004). These protocols are routine
procedures within the scope of one skilled in the art and from the
teaching herein.
[0167] The methods disclosed in the present specification include,
in part, introducing into a cell a polynucleotide molecule. A
polynucleotide molecule introduced into a cell can be transiently
or stably maintained by that cell. Stably-maintained polynucleotide
molecules may be extra-chromosomal and replicate autonomously, or
they may be integrated into the chromosomal material of the cell
and replicate non-autonomously. It is envisioned that any and all
methods for introducing a polynucleotide molecule disclosed in the
present specification into a cell can be used. Methods useful for
introducing a nucleic acid molecule into a cell include, without
limitation, chemical-mediated transfection such as, e.g., calcium
phosphate-mediated, diethyl-aminoethyl (DEAE) dextran-mediated,
lipid-mediated, polyethyleneimine (PEI)-mediated,
polylysine-mediated and polybrene-mediated; physical-mediated
tranfection, such as, e.g., biolistic particle delivery,
microinjection, protoplast fusion and electroporation; and
viral-mediated transfection, such as, e.g., retroviral-mediated
transfection, see, e.g., Introducing Cloned Genes into Cultured
Mammalian Cells, pp. 16.1-16.62 (Sambrook & Russell, eds.,
Molecular Cloning A Laboratory Manual, Vol. 3, 3.sup.rd ed. 2001).
One skilled in the art understands that selection of a specific
method to introduce an expression construct into a cell will
depend, in part, on whether the cell will transiently contain an
expression construct or whether the cell will stably contain an
expression construct. These protocols are routine procedures within
the scope of one skilled in the art and from the teaching
herein.
[0168] In an aspect of this embodiment, a chemical-mediated method,
termed transfection, is used to introduce a polynucleotide molecule
encoding a modified Clostridial toxin into a cell. In
chemical-mediated methods of transfection the chemical reagent
forms a complex with the nucleic acid that facilitates its uptake
into the cells. Such chemical reagents include, without limitation,
calcium phosphate-mediated, see, e.g., Martin Jordan & Florian
Worm, Transfection of Adherent and Suspended Cells by Calcium
Phosphate, 33(2) Methods 136-143 (2004); diethyl-aminoethyl (DEAE)
dextran-mediated, lipid-mediated, cationic polymer-mediated like
polyethyleneimine (PEI)-mediated and polylysine-mediated and
polybrene-mediated, see, e.g., Chun Zhang et al., Polyethylenimine
Strategies for Plasmid Delivery to Brain-Derived Cells, 33(2)
Methods 144-150 (2004). Such chemical-mediated delivery systems can
be prepared by standard methods and are commercially available,
see, e.g., CellPhect Transfection Kit (Amersham Biosciences,
Piscataway, N.J.); Mammalian Transfection Kit, Calcium phosphate
and DEAE Dextran, (Stratagene, Inc., La Jolla, Calif.);
Lipofectamine.TM. Transfection Reagent (Invitrogen, Inc., Carlsbad,
Calif.); ExGen 500 Transfection kit (Fermentas, Inc., Hanover,
Md.), and SuperFect and Effectene Transfection Kits (Qiagen, Inc.,
Valencia, Calif.).
[0169] In another aspect of this embodiment, a physical-mediated
method is used to introduce a polynucleotide molecule encoding a
modified Clostridial toxin into a cell. Physical techniques
include, without limitation, electroporation, biolistic and
microinjection. Biolistics and microinjection techniques perforate
the cell wall in order to introduce the nucleic acid molecule into
the cell, see, e.g., Jeike E. Biewenga et al., Plasmid-Mediated
Gene Transfer in Neurons Using the Biolistics Technique, 71(1) J.
Neurosci. Methods. 67-75 (1997); and John O'Brien & Sarah C. R.
Lummis, Biolistic and Diolistic Transfection: Using the Gene Gun to
Deliver DNA and Lipophilic Dyes into Mammalian Cells, 33(2) Methods
121-125 (2004). Electroporation, also termed
electropermeabilization, uses brief, high-voltage, electrical
pulses to create transient pores in the membrane through which the
nucleic acid molecules enter and can be used effectively for stable
and transient transfections of all cell types, see, e.g., M. Golzio
et al., In vitro and in vivo Electric Field-Mediated
Permeabilization, Gene Transfer, and Expression, 33(2) Methods
126-135 (2004); and Oliver Gresch et. al.., New Non-Viral Method
for Gene Transfer into Primary Cells, 33(2) Methods 151-163
(2004).
[0170] In another aspect of this embodiment, a viral-mediated
method, termed transduction, is used to introduce a polynucleotide
molecule encoding a modified Clostridial toxin into a cell. In
viral-mediated methods of transient transduction, the process by
which viral particles infect and replicate in a host cell has been
manipulated in order to use this mechanism to introduce a nucleic
acid molecule into the cell. Viral-mediated methods have been
developed from a wide variety of viruses including, without
limitation, retroviruses, adenoviruses, adeno-associated viruses,
herpes simplex viruses, picornaviruses, alphaviruses and
baculoviruses, see, e.g., Armin Blesch, Lentiviral and MLV based
Retroviral Vectors for ex vivo and in vivo Gene Transfer, 33(2)
Methods 164-172 (2004); and Maurizio Federico, From Lentiviruses to
Lentivirus Vectors, 229 Methods Mol. Biol. 3-15 (2003); E. M.
Poeschla, Non-Primate Lentiviral Vectors, 5(5) Curr. Opin. Mol.
Ther. 529-540 (2003); Karim Benihoud et al, Adenovirus Vectors for
Gene Delivery, 10(5) Curr. Opin. Biotechnol. 440-447 (1999); H.
Bueler, Adeno-Associated Viral Vectors for Gene Transfer and Gene
Therapy, 380(6) Biol. Chem. 613-622 (1999); Chooi M. Lai et al.,
Adenovirus and Adeno-Associated Virus Vectors, 21(12) DNA Cell
Biol. 895-913 (2002); Edward A. Burton et al., Gene Delivery Using
Herpes Simplex Virus Vectors, 21(12) DNA Cell Biol. 915-936 (2002);
Paola Grandi et al., Targeting HSV Amplicon Vectors, 33(2) Methods
179-186 (2004); Ilya Frolov et al., Alphavirus-Based Expression
Vectors: Strategies and Applications, 93(21) Proc. Natl. Acad. Sci.
U.S.A. 11371-11377 (1996); Markus U. Ehrengruber, Alphaviral Gene
Transfer in Neurobiology, 59(1) Brain Res. Bull. 13-22 (2002);
Thomas A. Kost & J. Patrick Condreay, Recombinant Baculoviruses
as Mammalian Cell Gene-Delivery Vectors, 20(4) Trends Biotechnol.
173-180 (2002); and A. Huser & C. Hofmann, Baculovirus Vectors:
Novel Mammalian Cell Gene-Delivery Vehicles and Their Applications,
3(1) Am. J. Pharmacogenomics 53-63 (2003).
[0171] Adenoviruses, which are non-enveloped, double-stranded DNA
viruses, are often selected for mammalian cell transduction because
adenoviruses handle relatively large polynucleotide molecules of
about 36 kb, are produced at high titer, and can efficiently infect
a wide variety of both dividing and non-dividing cells, see, e.g.,
Wim T. J. M. C. Hermens et al., Transient Gene Transfer to Neurons
and Glia: Analysis of Adenoviral Vector Performance in the CNS and
PNS, 71(1) J. Neurosci. Methods 85-98 (1997); and Hiroyuki
Mizuguchi et al., Approaches for Generating Recombinant Adenovirus
Vectors, 52(3) Adv. Drug Deliv. Rev. 165-176 (2001). Transduction
using adenoviral-based system do not support prolonged protein
expression because the nucleic acid molecule is carried from an
episome in the cell nucleus, rather than being integrated into the
host cell chromosome. Adenoviral vector systems and specific
protocols for how to use such vectors are disclosed in, e.g.,
ViraPower.TM. Adenoviral Expression System (Invitrogen, Inc.,
Carlsbad, Calif.) and ViraPower.TM. Adenoviral Expression System
Instruction Manual 25-0543 version A, Invitrogen, Inc., (Jul. 15,
2002); and AdEasy.TM. Adenoviral Vector System (Stratagene, Inc.,
La Jolla, Calif.) and AdEasy.TM. Adenoviral Vector System
Instruction Manual 064004f, Stratagene, Inc.
[0172] Polynucleotide molecule delivery can also use
single-stranded RNA retroviruses, such as, e.g., oncoretroviruses
and lentiviruses. Retroviral-mediated transduction often produce
transduction efficiencies close to 100%, can easily control the
proviral copy number by varying the multiplicity of infection
(MOI), and can be used to either transiently or stably transduce
cells, see, e.g., Tiziana Tonini et al., Transient Production Of
Retroviral-and Lentiviral-Based Vectors For the Transduction of
Mammalian Cells, 285 Methods Mol. Biol. 141-148 (2004); Armin
Blesch, Lentiviral and MLV Based Retroviral Vectors for ex vivo and
in vivo Gene Transfer, 33(2) Methods 164-172 (2004); Felix
Recillas-Targa, Gene Transfer and Expression in Mammalian Cell
Lines and Transgenic Animals, 267 Methods Mol. Biol. 417-433
(2004); and Roland Wolkowicz et al., Lentiviral Vectors for the
Delivery of DNA into Mammalian Cells, 246 Methods Mol. Biol.
391-411 (2004). Retroviral particles consist of an RNA genome
packaged in a protein capsid, surrounded by a lipid envelope. The
retrovirus infects a host cell by injecting its RNA into the
cytoplasm along with the reverse transcriptase enzyme. The RNA
template is then reverse transcribed into a linear, double stranded
cDNA that replicates itself by integrating into the host cell
genome. Viral particles are spread both vertically (from parent
cell to daughter cells via the provirus) as well as horizontally
(from cell to cell via virions). This replication strategy enables
long-term persistent expression since the nucleic acid molecules of
interest are stably integrated into a chromosome of the host cell,
thereby enabling long-term expression of the protein. For instance,
animal studies have shown that lentiviral vectors injected into a
variety of tissues produced sustained protein expression for more
than 1 year, see, e.g., Luigi Naldini et al., In vivo Gene Delivery
and Stable Transduction of Non-Dividing Cells By a Lentiviral
Vector, 272(5259) Science 263-267 (1996). The
Oncoretroviruses-derived vector systems, such as, e.g., Moloney
murine leukemia virus (MoMLV), are widely used and infect many
different non-dividing cells. Lentiviruses can also infect many
different cell types, including dividing and non-dividing cells and
possess complex envelope proteins, which allows for highly specific
cellular targeting.
[0173] Retroviral vectors and specific protocols for how to use
such vectors are disclosed in, e.g., U.S. patent Nos. Manfred
Gossen & Hermann Bujard, Tight Control of Gene Expression in
Eukaryotic Cells By Tetracycline-Responsive Promoters, U.S. Pat.
No. 5,464,758 (Nov. 7, 1995) and Hermann Bujard & Manfred
Gossen, Methods for Regulating Gene Expression, U.S. Pat. No.
5,814,618 (Sep. 29, 1998) David S. Hogness, Polynucleotides
Encoding Insect Steroid Hormone Receptor Polypeptides and Cells
Transformed With Same, U.S. Pat. No. 5,514,578 (May 7, 1996) and
David S. Hogness, Polynucleotide Encoding Insect Ecdysone Receptor,
U.S. Pat. No. 6,245,531 (Jun. 12, 2001); Elisabetta Vegeto et al.,
Progesterone Receptor Having C. Terminal Hormone Binding Domain
Truncations, U.S. Pat. No. 5,364,791 (Nov. 15, 1994), Elisabetta
Vegeto et al., Mutated Steroid Hormone Receptors, Methods For Their
Use and Molecular Switch For Gene Therapy, U.S. Pat. No. 5,874,534
(Feb. 23, 1999) and Elisabetta Vegeto et al., Mutated Steroid
Hormone Receptors, Methods For Their Use and Molecular Switch For
Gene Therapy, U.S. Pat. No. 5,935,934 (Aug. 10, 1999). Furthermore,
such viral delivery systems can be prepared by standard methods and
are commercially available, see, e.g., BD.TM. Tet-Off and Tet-On
Gene Expression Systems (BD Biosciences-Clonetech, Palo Alto,
Calif.) and BD.TM. Tet-Off and Tet-On Gene Expression Systems User
Manual, PT3001-1, BD Biosciences Clonetech, (Mar. 14, 2003),
GeneSwitch.TM. System (Invitrogen, Inc., Carlsbad, Calif.) and
GeneSwitch.TM. System A Mifepristone-Regulated Expression System
for Mammalian Cells version D, 25-0313, Invitrogen, Inc., (Nov. 4,
2002); ViraPower.TM. Lentiviral Expression System (Invitrogen,
Inc., Carlsbad, Calif.) and ViraPower.TM. Lentiviral Expression
System Instruction Manual 25-0501 version E, Invitrogen, Inc.,
(Dec. 8, 2003); and Complete Control.RTM. Retroviral Inducible
Mammalian Expression System (Stratagene, La Jolla, Calif.) and
Complete Control.RTM. Retroviral Inducible Mammalian Expression
System Instruction Manual, 064005e.
[0174] The methods disclosed in the present specification include,
in part, expressing a modified Clostridial toxin from a
polynucleotide molecule. It is envisioned that any of a variety of
expression systems may be useful for expressing a modified
Clostridial toxin from a polynucleotide molecule disclosed in the
present specification, including, without limitation, cell-based
systems and cell-free expression systems. Cell-based systems
include, without limitation, viral expression systems, prokaryotic
expression systems, yeast expression systems, baculoviral
expression systems, insect expression systems and mammalian
expression systems. Cell-free systems include, without limitation,
wheat germ extracts, rabbit reticulocyte extracts and E. coli
extracts and generally are equivalent to the method disclosed
herein. Expression of a polynucleotide molecule using an expression
system can include any of a variety of characteristics including,
without limitation, inducible expression, non-inducible expression,
constitutive expression, viral-mediated expression,
stably-integrated expression, and transient expression. Expression
systems that include well-characterized vectors, reagents,
conditions and cells are well-established and are readily available
from commercial vendors that include, without limitation, Ambion,
Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.; BD
Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc,
Carlsbad, Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied
Science, Indianapolis, Ind.; and Stratagene, La Jolla, Calif.
Non-limiting examples on the selection and use of appropriate
heterologous expression systems are described in e.g., PROTEIN
EXPRESSION. A PRACTICAL APPROACH (S. J. Higgins and B. David Hames
eds., Oxford University Press, 1999); Joseph M. Fernandez &
James P. Hoeffler, GENE EXPRESSION SYSTEMS. USING NATURE FOR THE
ART OF EXPRESSION (Academic Press, 1999); and Meena Rai &
Harish Padh, Expression Systems for Production of Heterologous
Proteins, 80(9) Curr. Sci. 1121-1128, (2001). These protocols are
routine procedures well within the scope of one skilled in the art
and from the teaching herein.
[0175] A variety of cell-based expression procedures are useful for
expressing a modified Clostridial toxin encoded by polynucleotide
molecule disclosed in the present specification. Examples included,
without limitation, viral expression systems, prokaryotic
expression systems, yeast expression systems, baculoviral
expression systems, insect expression systems and mammalian
expression systems. Viral expression systems include, without
limitation, the ViraPower.TM. Lentiviral (Invitrogen, Inc.,
Carlsbad, Calif.), the Adenoviral Expression Systems (Invitrogen,
Inc., Carlsbad, Calif.), the AdEasy.TM. XL Adenoviral Vector System
(Stratagene, La Jolla, Calif.) and the ViraPort.RTM. Retroviral
Gene Expression System (Stratagene, La Jolla, Calif.). Non-limiting
examples of prokaryotic expression systems include the Champion.TM.
pET Expression System (EMD Biosciences-Novagen, Madison, Wis.), the
TriEx.TM. Bacterial Expression Systems (EMD Biosciences-Novagen,
Madison, Wis.), the QIAexpress.RTM. Expression System (QIAGEN,
Inc.), and the Affinity.RTM. Protein Expression and Purification
System (Stratagene, La Jolla, Calif.). Yeast expression systems
include, without limitation, the EasySelect.TM. Pichia Expression
Kit (Invitrogen, Inc., Carlsbad, Calif.), the YES-Echo.TM.
Expression Vector Kits (Invitrogen, Inc., Carlsbad, Calif.) and the
SpECTRA.TM. S. pombe Expression System (Invitrogen, Inc., Carlsbad,
Calif.). Non-limiting examples of baculoviral expression systems
include the BaculoDirect.TM. (Invitrogen, Inc., Carlsbad, Calif.),
the Bac-to-Bac.RTM. (Invitrogen, Inc., Carlsbad, Calif.), and the
BD BaculoGold.TM. (BD Biosciences-Pharmigen, San Diego, Calif.).
Insect expression systems include, without limitation, the
Drosophila Expression System (DES.RTM.) (Invitrogen, Inc.,
Carlsbad, Calif.), InsectSelect.TM. System (Invitrogen, Inc.,
Carlsbad, Calif.) and InsectDirect.TM. System (EMD
Biosciences-Novagen, Madison, Wis.). Non-limiting examples of
mammalian expression systems include the T-REx.TM.
(Tetracycline-Regulated Expression) System (Invitrogen, Inc.,
Carlsbad, Calif.), the FIp-In.TM. T-REx.TM. System (Invitrogen,
Inc., Carlsbad, Calif.), the pcDNA.TM. system (Invitrogen, Inc.,
Carlsbad, Calif.), the pSecTag2 system (Invitrogen, Inc., Carlsbad,
Calif.), the Exchanger.RTM. System, InterPlay.TM. Mammalian TAP
System (Stratagene, La Jolla, Calif.), Complete Control.RTM.
Inducible Mammalian Expression System (Stratagene, La Jolla,
Calif.) and LacSwitch.RTM. II Inducible Mammalian Expression System
(Stratagene, La Jolla, Calif.).
[0176] Another procedure of expressing a modified Clostridial toxin
encoded by polynucleotide molecule disclosed in the present
specification employs a cell-free expression system such as,
without limitation, prokaryotic extracts and eukaryotic extracts.
Non-limiting examples of prokaryotic cell extracts include the RTS
100 E. coli HY Kit (Roche Applied Science, Indianapolis, Ind.), the
ActivePro In Vitro Translation Kit (Ambion, Inc., Austin, Tex.),
the EcoPro.TM. System (EMD Biosciences-Novagen, Madison, Wis.) and
the Expressway.TM. Plus Expression System (Invitrogen, Inc.,
Carlsbad, Calif.). Eukaryotic cell extract include, without
limitation, the RTS 100 Wheat Germ CECF Kit (Roche Applied Science,
Indianapolis, Ind.), the TnT.RTM. Coupled Wheat Germ Extract
Systems (Promega Corp., Madison, Wis.), the Wheat Germ IVT.TM. Kit
(Ambion, Inc., Austin, Tex.), the Retic Lysate IVT.TM. Kit (Ambion,
Inc., Austin, Tex.), the PROTEINscript.RTM. II System (Ambion,
Inc., Austin, Tex.) and the TnT.RTM. Coupled Reticulocyte Lysate
Systems (Promega Corp., Madison, Wis.).
[0177] Another aspect of the present invention provides a method of
activating a modified Clostridial toxin comprising an exogenous
Clostridial toxin di-chain loop region including a Clostridial
toxin di-chain loop protease cleavage site from a different
Clostridial toxin, such method comprising the step of incubating
the modified Clostridial toxin with a Clostridial toxin di-chain
loop protease under physiological conditions, wherein the
Clostridial toxin di-chain loop protease is capable of cleaving the
Clostridial toxin di-chain loop protease cleavage site present in
the exogenous Clostridial toxin di-chain loop region and wherein
cleavage of the modified Clostridial toxin by the Clostridial toxin
di-chain loop protease converts the modified Clostridial toxin from
its single-chain polypeptide form into its di-chain form, thereby
activating the modified Clostridial toxin.
[0178] Another aspect of the present invention provides a method of
activating a recombinantly-expressed Clostridial toxin, such method
comprising the step of incubating the Clostridial toxin with a
Clostridial toxin di-chain loop protease under physiological
conditions, wherein the Clostridial toxin di-chain loop protease is
capable of cleaving the Clostridial toxin di-chain loop protease
cleavage site present in the Clostridial toxin di-chain loop region
and wherein cleavage of the Clostridial toxin by the Clostridial
toxin di-chain loop protease converts the Clostridial toxin from
its single-chain polypeptide form into its di-chain form, thereby
activating the recombinantly-expressed Clostridial toxin.
[0179] Aspects of the present invention provide, in part, a
Clostridial toxin di-chain loop protease. As used herein, the term
"Clostridial toxin di-chain loop protease" means any protease
capable of selectively cleaving the P.sub.1--P.sub.1' scissile bond
comprising the di-chain loop protease cleavage site. As used
herein, the term "selectively" means having a highly preferred
activity or effect. Thus, with reference to a Clostridial toxin
di-chain loop protease, there is a discriminatory proteolytic
cleavage of the P.sub.1--P.sub.1' scissile bond comprising the
di-chain loop protease cleavage site. It is envisioned that any and
all proteases capable of selectively cleaving the P.sub.1--P.sub.1'
scissile bond comprising the di-chain loop protease cleavage site
can be useful in the disclosed methods, including, without
exception, a sulfhydryl proteinase. One example of a sulfhydryl
proteinase is clostripain, also known as clostridiopeptidase B,
endoproteinase-Arg-C, or .gamma.-protease. See, e.g., William M.
Mitchell & William F. Harrington, Purification and Properties
of Clostridiopeptidase B (Clostripain), 243(18) J. Biol. Chem.
4683-4692. (1968); William M. Mitchell & William F. Harrington,
Clostripain, 19 Methods Enzymol. 635-642 (1970); and Ashu A.
Kembhavi, et al., Clostripain: Characterization of the Active Site,
283(2) FEBS Lett. 277-280 (1991), each of which is hereby
incorporated by reference in its entirety. This two chain cysteine
proteinase is highly specific for the carboxyl peptide bond of
arginine. Non-limiting examples of clostripain include SEQ ID NO:
33, SEQ ID NO; 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and
SEQ ID NO: 38. Clostripain selectively hydrolysis of arginyl bonds,
although lysyl bonds are cleaved at a lower rate.
[0180] A clostripain useful in aspects of the invention includes,
without limitation, naturally occurring clostripain; naturally
occurring clostripain variants; and non-naturally-occurring
clostripain variants, such as, e.g., conservative clostripain
variants, non-conservative clostripain variants and clostripain
peptidomimetics. As used herein, the term "clostripain variant,"
whether naturally-occurring or non-naturally-occurring, means a
clostripain that has at least one amino acid change from the
corresponding region of the disclosed reference sequences and can
be described in percent identity to the corresponding region of
that reference sequence. Any of a variety of sequence alignment
methods can be used to determine percent identity, including,
without limitation, global methods, local methods and hybrid
methods, such as, e.g., segment approach methods. Protocols to
determine percent identity are routine procedures within the scope
of one skilled in the art and from the teaching herein.
[0181] As used herein, the term "naturally occurring clostripain
variant" means any clostripain produced without the aid of any
human manipulation, including, without limitation, clostripain
isoforms produced from alternatively-spliced transcripts,
clostripain isoforms produced by spontaneous mutation and
clostripain subtypes. Non-limiting examples of a clostripain
isoform include, e.g., BoNT/A di-chain loop region isoforms, BoNT/B
di-chain loop region isoforms, BoNT/C1 di-chain loop region
isoforms, BoNT/D di-chain loop region isoforms, BoNT/E di-chain
loop region isoforms, BoNT/F di-chain loop region isoforms, BoNT/G
di-chain loop region isoforms, TeNT di-chain loop region isoforms,
BaNT di-chain loop region isoforms, and BuNT di-chain loop region
isoforms. Non-limiting examples of a Clostridial toxin subtype
include, e.g., BoNT/A di-chain loop region subtypes such as, e.g.,
a BoNT/A1 di-chain loop region, a BoNT/A2 di-chain loop region, a
BoNT/A3 di-chain loop region and a BoNT/A4 di-chain loop region;
BoNT/B di-chain loop region subtypes, such as, e.g., a BoNT/B1
di-chain loop region, a BoNT/B2 di-chain loop region, a BoNT/B
bivalent di-chain loop region and a BoNT/B nonproteolytic di-chain
loop region; BoNT/C1 di-chain loop region subtypes, such as, e.g.,
a BoNT/C1-1 di-chain loop region and a BoNT/C1-2 di-chain loop
region; BoNT/E di-chain loop region subtypes, such as, e.g., a
BoNT/E1 di-chain loop region, a BoNT/E2 di-chain loop region and a
BoNT/E3 di-chain loop region; and BoNT/F di-chain loop region
subtypes, such as, e.g., a BoNT/F1 di-chain loop region, a BoNT/F2
di-chain loop region, a BoNT/F3 di-chain loop region and a BoNT/F4
di-chain loop region.
[0182] As used herein, the term "non-naturally occurring
clostripain variant" means any clostripain produced with the aid of
human manipulation, including, without limitation, clostripain
variants produced by genetic engineering using random mutagenesis
or rational design and clostripain variants produced by chemical
synthesis. Non-limiting examples of non-naturally occurring
clostripain variants include, e.g., conservative clostripain
variants, non-conservative clostripain variants and clostripain
peptidomimetics.
[0183] As used herein, the term "conservative clostripain variant"
means a clostripain that has at least one amino acid substituted by
another amino acid or an amino acid analog that has at least one
property similar to that of the original amino acid from the
reference clostripain sequence. Examples of properties include,
without limitation, similar size, topography, charge,
hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding
capacity, hydrogen-bonding capacity, a physicochemical property, of
the like, or any combination thereof. A conservative clostripain
variant can function in substantially the same manner as the
reference clostripain on which the conservative clostripain variant
is based, and can be substituted for the reference clostripain in
any aspect of the present invention. A conservative clostripain
variant may substitute one or more amino acids, two or more amino
acids, three or more amino acids, four or more amino acids or five
or more amino acids from the reference clostripain on which the
conservative clostripain variant is based. A conservative
clostripain variant can also possess at least 50% amino acid
identity, 65% amino acid identity, 75% amino acid identity, 85%
amino acid identity or 95% amino acid identity to the reference
clostripain on which the conservative clostripain variant is based.
Non-limiting examples of a conservative clostripain variant
include, e.g., conservative clostripain variants of SEQ ID NO: 33,
conservative clostripain variants of SEQ ID NO: 34, conservative
clostripain variants of SEQ ID NO: 35, conservative clostripain
variants of SEQ ID NO: 36, conservative clostripain variants of SEQ
ID NO: 37, and conservative clostripain variants of SEQ ID NO:
38.
[0184] As used herein, the term "non-conservative clostripain
variant" means a clostripain in which 1) at least one amino acid is
deleted from the reference clostripain on which the
non-conservative clostripain variant is based; 2) at least one
amino acid added to the reference clostripain on which the
non-conservative clostripain is based; or 3) at least one amino
acid is substituted by another amino acid or an amino acid analog
that does not share any property similar to that of the original
amino acid from the reference clostripain sequence. A
non-conservative clostripain variant can function in substantially
the same manner as the reference clostripain on which the
non-conservative clostripain is based, and can be substituted for
the reference clostripain in any aspect of the present invention. A
non-conservative clostripain variant can add one or more amino
acids, two or more amino acids, three or more amino acids, four or
more amino acids, five or more amino acids, and ten or more amino
acids to the reference clostripain on which the non-conservative
clostripain variant is based. A non-conservative clostripain may
substitute one or more amino acids, two or more amino acids, three
or more amino acids, four or more amino acids or five or more amino
acids from the reference clostripain on which the non-conservative
clostripain variant is based. A non-conservative clostripain
variant can also possess at least 50% amino acid identity, 65%
amino acid identity, 75% amino acid identity, 85% amino acid
identity or 95% amino acid identity to the reference clostripain on
which the non-conservative clostripain variant is based.
Non-limiting examples of a non-conservative clostripain variant
include, e.g., non-conservative clostripain variants of SEQ ID NO:
33, non-conservative clostripain variants of SEQ ID NO: 34,
non-conservative clostripain variants of SEQ ID NO: 35,
non-conservative clostripain variants of SEQ ID NO: 36,
non-conservative clostripain variants of SEQ ID NO: 37, and
non-conservative clostripain variants of SEQ ID NO: 38.
[0185] As used herein, the term "clostripain peptidomimetic" means
a clostripain that has at least one amino acid substituted by a
non-natural oligomer that has at least one property similar to that
of the first amino acid. Examples of properties include, without
limitation, topography of a peptide primary structural element,
functionality of a peptide primary structural element, topology of
a peptide secondary structural element, functionality of a peptide
secondary structural element, of the like, or any combination
thereof. A clostripain peptidomimetic can function in substantially
the same manner as the reference clostripain on which the
clostripain peptidomimetic is based, and can be substituted for the
reference clostripain in any aspect of the present invention. A
clostripain peptidomimetic may substitute one or more amino acids,
two or more amino acids, three or more amino acids, four or more
amino acids or five or more amino acids from the reference
clostripain on which the clostripain peptidomimetic is based. A
clostripain peptidomimetic can also possess at least 50% amino acid
identity, at least 65% amino acid identity, at least 75% amino acid
identity, at least 85% amino acid identity or at least 95% amino
acid identity to the reference clostripain on which the clostripain
peptidomimetic is based. For examples of peptidomimetic methods
see, e.g., Amy S. Ripka & Daniel H. Rich, Peptidomimetic
design, 2(4) CURR. OPIN. CHEM. BIOL. 441-452 (1998); and M. Angels
Estiarte & Daniel H. Rich, Peptidomimetics for Drug Design,
803-861 (BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY Vol. 1
PRINCIPLE AND PRACTICE, Donald J. Abraham ed., Wiley-Interscience,
6.sup.th ed 2003). Non-limiting examples of a clostripain
peptidomimetic include, e.g., clostripain peptidomimetics of SEQ ID
NO: 33, clostripain peptidomimetics of SEQ ID NO: 34, clostripain
peptidomimetics of SEQ ID NO: 35, clostripain peptidomimetics of
SEQ ID NO: 36, clostripain peptidomimetics of SEQ ID NO: 37, and
clostripain peptidomimetics of SEQ ID NO: 38.
[0186] Thus, in an embodiment, a Clostridial toxin di-chain loop
protease comprises a clostripain. In an aspect of this embodiment,
a clostripain can be a naturally occurring clostripain variant,
such as, e.g., a clostripain isoform or a clostripain subtype. In
another aspect of this embodiment, a clostripain can be a
non-naturally occurring clostripain variant, such as, e.g., a
conservative clostripain variant, a non-conservative clostripain
variant or an active clostripain fragment, or any combination
thereof.
[0187] In another embodiment, a clostripain comprises a naturally
occurring clostripain variant of SEQ ID NO: 33, such as, e.g., a
clostripain isoform of SEQ ID NO: 33 or a clostripain subtype of
SEQ ID NO: 33. In still another aspect of this embodiment, a
clostripain comprises a non-naturally occurring clostripain variant
of SEQ ID NO: 33, such as, e.g., a conservative clostripain variant
of SEQ ID NO: 33, a non-conservative clostripain variant of SEQ ID
NO: 33 or an active clostripain fragment of SEQ ID NO: 33, or any
combination thereof. In yet another embodiment, a clostripain
comprises a clostripain of SEQ ID NO: 33.
[0188] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at least 70% amino acid identity with
SEQ ID NO: 33, at least 75% amino acid identity with SEQ ID NO: 33,
at least 80% amino acid identity with SEQ ID NO: 33, at least 85%
amino acid identity with SEQ ID NO: 33, at least 90% amino acid
identity with SEQ ID NO: 33 or at least 95% amino acid identity
with SEQ ID NO: 33. In yet other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at most 70% amino
acid identity with SEQ ID NO: 33, at most 75% amino acid identity
with SEQ ID NO: 33, at most 80% amino acid identity with SEQ ID NO:
33, at most 85% amino acid identity with SEQ ID NO: 33, at most 90%
amino acid identity with SEQ ID NO: 33 or at most 95% amino acid
identity with SEQ ID NO: 33.
[0189] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid substitutions relative to SEQ ID NO: 33. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
substitutions relative to SEQ ID NO: 33. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions
relative to SEQ ID NO: 33. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions relative to SEQ ID
NO: 33. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid additions relative to SEQ ID NO: 33. In
other aspects of this embodiment, a clostripain comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid additions relative to SEQ ID NO: 33.
[0190] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 contiguous
amino acid substitutions relative to SEQ ID NO: 33. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
substitutions relative to SEQ ID NO: 33. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 contiguous amino acid deletions relative
to SEQ ID NO: 33. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions relative to SEQ ID NO:
33. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
contiguous amino acid additions relative to SEQ ID NO: 33. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
additions relative to SEQ ID NO: 33.
[0191] In another embodiment, a clostripain comprises a naturally
occurring clostripain variant of SEQ ID NO: 34, such as, e.g., a
clostripain isoform of SEQ ID NO: 34 or a clostripain subtype of
SEQ ID NO: 34. In still another aspect of this embodiment, a
clostripain comprises a non-naturally occurring clostripain variant
of SEQ ID NO: 34, such as, e.g., a conservative clostripain variant
of SEQ ID NO: 34, a non-conservative clostripain variant of SEQ ID
NO: 34 or an active clostripain fragment of SEQ ID NO: 34, or any
combination thereof. In yet another embodiment, a clostripain
comprises a clostripain of SEQ ID NO: 34.
[0192] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at least 70% amino acid identity with
SEQ ID NO: 34, at least 75% amino acid identity with SEQ ID NO: 34,
at least 80% amino acid identity with SEQ ID NO: 34, at least 85%
amino acid identity with SEQ ID NO: 34, at least 90% amino acid
identity with SEQ ID NO: 34 or at least 95% amino acid identity
with SEQ ID NO: 34. In yet other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at most 70% amino
acid identity with SEQ ID NO: 34, at most 75% amino acid identity
with SEQ ID NO: 34, at most 80% amino acid identity with SEQ ID NO:
34, at most 85% amino acid identity with SEQ ID NO: 34, at most 90%
amino acid identity with SEQ ID NO: 34 or at most 95% amino acid
identity with SEQ ID NO: 34.
[0193] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid substitutions relative to SEQ ID NO: 34. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
substitutions relative to SEQ ID NO: 34. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions
relative to SEQ ID NO: 34. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions relative to SEQ ID
NO: 34. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid additions relative to SEQ ID NO: 34. In
other aspects of this embodiment, a clostripain comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid additions relative to SEQ ID NO: 34.
[0194] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 contiguous
amino acid substitutions relative to SEQ ID NO: 34. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
substitutions relative to SEQ ID NO: 34. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 contiguous amino acid deletions relative
to SEQ ID NO: 34. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions relative to SEQ ID NO:
34. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
contiguous amino acid additions relative to SEQ ID NO: 34. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
additions relative to SEQ ID NO: 34.
[0195] In another embodiment, a clostripain comprises a naturally
occurring clostripain variant of SEQ ID NO: 35, such as, e.g., a
clostripain isoform of SEQ ID NO: 35 or a clostripain subtype of
SEQ ID NO: 35. In still another aspect of this embodiment, a
clostripain comprises a non-naturally occurring clostripain variant
of SEQ ID NO: 35, such as, e.g., a conservative clostripain variant
of SEQ ID NO: 35, a non-conservative clostripain variant of SEQ ID
NO: 35 or an active clostripain fragment of SEQ ID NO: 35, or any
combination thereof. In yet another embodiment, a clostripain
comprises a clostripain of SEQ ID NO: 35.
[0196] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at least 70% amino acid identity with
SEQ ID NO: 35, at least 75% amino acid identity with SEQ ID NO: 35,
at least 80% amino acid identity with SEQ ID NO: 35, at least 85%
amino acid identity with SEQ ID NO: 35, at least 90% amino acid
identity with SEQ ID NO: 35 or at least 95% amino acid identity
with SEQ ID NO: 35. In yet other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at most 70% amino
acid identity with SEQ ID NO: 35, at most 75% amino acid identity
with SEQ ID NO: 35, at most 80% amino acid identity with SEQ ID NO:
35, at most 85% amino acid identity with SEQ ID NO: 35, at most 90%
amino acid identity with SEQ ID NO: 35 or at most 95% amino acid
identity with SEQ ID NO: 35.
[0197] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid substitutions relative to SEQ ID NO: 35. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
substitutions relative to SEQ ID NO: 35. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions
relative to SEQ ID NO: 35. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions relative to SEQ ID
NO: 35. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid additions relative to SEQ ID NO: 35. In
other aspects of this embodiment, a clostripain comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid additions relative to SEQ ID NO: 35.
[0198] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 contiguous
amino acid substitutions relative to SEQ ID NO: 35. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
substitutions relative to SEQ ID NO: 35. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 contiguous amino acid deletions relative
to SEQ ID NO: 35. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions relative to SEQ ID NO:
35. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
contiguous amino acid additions relative to SEQ ID NO: 35. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
additions relative to SEQ ID NO: 35.
[0199] In another embodiment, a clostripain comprises a naturally
occurring clostripain variant of SEQ ID NO: 36, such as, e.g., a
clostripain isoform of SEQ ID NO: 36 or a clostripain subtype of
SEQ ID NO: 36. In still another aspect of this embodiment, a
clostripain comprises a non-naturally occurring clostripain variant
of SEQ ID NO: 36, such as, e.g., a conservative clostripain variant
of SEQ ID NO: 36, a non-conservative clostripain variant of SEQ ID
NO: 36 or an active clostripain fragment of SEQ ID NO: 36, or any
combination thereof. In yet another embodiment, a clostripain
comprises a clostripain of SEQ ID NO: 36.
[0200] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at least 70% amino acid identity with
SEQ ID NO: 36, at least 75% amino acid identity with SEQ ID NO: 36,
at least 80% amino acid identity with SEQ ID NO: 36, at least 85%
amino acid identity with SEQ ID NO: 36, at least 90% amino acid
identity with SEQ ID NO: 36 or at least 95% amino acid identity
with SEQ ID NO: 36. In yet other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at most 70% amino
acid identity with SEQ ID NO: 36, at most 75% amino acid identity
with SEQ ID NO: 36, at most 80% amino acid identity with SEQ ID NO:
36, at most 85% amino acid identity with SEQ ID NO: 36, at most 90%
amino acid identity with SEQ ID NO: 36 or at most 95% amino acid
identity with SEQ ID NO: 36.
[0201] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid substitutions relative to SEQ ID NO: 36. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
substitutions relative to SEQ ID NO: 36. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions
relative to SEQ ID NO: 36. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions relative to SEQ ID
NO: 36. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid additions relative to SEQ ID NO: 36. In
other aspects of this embodiment, a clostripain comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid additions relative to SEQ ID NO: 36.
[0202] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 contiguous
amino acid substitutions relative to SEQ ID NO: 36. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
substitutions relative to SEQ ID NO: 36. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 contiguous amino acid deletions relative
to SEQ ID NO: 36. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions relative to SEQ ID NO:
36. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
contiguous amino acid additions relative to SEQ ID NO: 36. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
additions relative to SEQ ID NO: 36.
[0203] In another embodiment, a clostripain comprises a naturally
occurring clostripain variant of SEQ ID NO: 37, such as, e.g., a
clostripain isoform of SEQ ID NO: 37 or a clostripain subtype of
SEQ ID NO: 37. In still another aspect of this embodiment, a
clostripain comprises a non-naturally occurring clostripain variant
of SEQ ID NO: 37, such as, e.g., a conservative clostripain variant
of SEQ ID NO: 37, a non-conservative clostripain variant of SEQ ID
NO: 37 or an active clostripain fragment of SEQ ID NO: 37, or any
combination thereof. In yet another embodiment, a clostripain
comprises a clostripain of SEQ ID NO: 37.
[0204] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at least 70% amino acid identity with
SEQ ID NO: 37, at least 75% amino acid identity with SEQ ID NO: 37,
at least 80% amino acid identity with SEQ ID NO: 37, at least 85%
amino acid identity with SEQ ID NO: 37, at least 90% amino acid
identity with SEQ ID NO: 37 or at least 95% amino acid identity
with SEQ ID NO: 37. In yet other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at most 70% amino
acid identity with SEQ ID NO: 37, at most 75% amino acid identity
with SEQ ID NO: 37, at most 80% amino acid identity with SEQ ID NO:
37, at most 85% amino acid identity with SEQ ID NO: 37, at most 90%
amino acid identity with SEQ ID NO: 37 or at most 95% amino acid
identity with SEQ ID NO: 37.
[0205] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid substitutions relative to SEQ ID NO: 37. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
substitutions relative to SEQ ID NO: 37. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions
relative to SEQ ID NO: 37. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions relative to SEQ ID
NO: 37. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid additions relative to SEQ ID NO: 37. In
other aspects of this embodiment, a clostripain comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid additions relative to SEQ ID NO: 37.
[0206] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 contiguous
amino acid substitutions relative to SEQ ID NO: 37. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
substitutions relative to SEQ ID NO: 37. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 contiguous amino acid deletions relative
to SEQ ID NO: 37. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions relative to SEQ ID NO:
37. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
contiguous amino acid additions relative to SEQ ID NO: 37. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
additions relative to SEQ ID NO: 37.
[0207] In another embodiment, a clostripain comprises a naturally
occurring clostripain variant of SEQ ID NO: 38, such as, e.g., a
clostripain isoform of SEQ ID NO: 38 or a clostripain subtype of
SEQ ID NO: 38. In still another aspect of this embodiment, a
clostripain comprises a non-naturally occurring clostripain variant
of SEQ ID NO: 38, such as, e.g., a conservative clostripain variant
of SEQ ID NO: 38, a non-conservative clostripain variant of SEQ ID
NO: 38 or an active clostripain fragment of SEQ ID NO: 38, or any
combination thereof. In yet another embodiment, a clostripain
comprises a clostripain of SEQ ID NO: 38.
[0208] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at least 70% amino acid identity with
SEQ ID NO: 38, at least 75% amino acid identity with SEQ ID NO: 38,
at least 80% amino acid identity with SEQ ID NO: 38, at least 85%
amino acid identity with SEQ ID NO: 38, at least 90% amino acid
identity with SEQ ID NO: 38 or at least 95% amino acid identity
with SEQ ID NO: 38. In yet other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at most 70% amino
acid identity with SEQ ID NO: 38, at most 75% amino acid identity
with SEQ ID NO: 38, at most 80% amino acid identity with SEQ ID NO:
38, at most 85% amino acid identity with SEQ ID NO: 38, at most 90%
amino acid identity with SEQ ID NO: 38 or at most 95% amino acid
identity with SEQ ID NO: 38.
[0209] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid substitutions relative to SEQ ID NO: 38. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous amino acid
substitutions relative to SEQ ID NO: 38. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 non-contiguous amino acid deletions
relative to SEQ ID NO: 38. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 non-contiguous amino acid deletions relative to SEQ ID
NO: 38. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
non-contiguous amino acid additions relative to SEQ ID NO: 38. In
other aspects of this embodiment, a clostripain comprises a
polypeptide having, e.g., at least one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 non-contiguous
amino acid additions relative to SEQ ID NO: 38.
[0210] In other aspects of this embodiment, a clostripain comprises
a polypeptide having, e.g., at most one, two, three, four, five,
six, seven, eight, nine, 10, 20, 30, 40, 50, or 100 contiguous
amino acid substitutions relative to SEQ ID NO: 38. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
substitutions relative to SEQ ID NO: 38. In yet other aspects of
this embodiment, a clostripain comprises a polypeptide having,
e.g., at most one, two, three, four, five, six, seven, eight, nine,
10, 20, 30, 40, 50, or 100 contiguous amino acid deletions relative
to SEQ ID NO: 38. In other aspects of this embodiment, a
clostripain comprises a polypeptide having, e.g., at least one,
two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40,
50, or 100 contiguous amino acid deletions relative to SEQ ID NO:
38. In still other aspects of this embodiment, a clostripain
comprises a polypeptide having, e.g., at most one, two, three,
four, five, six, seven, eight, nine, 10, 20, 30, 40, 50, or 100
contiguous amino acid additions relative to SEQ ID NO: 38. In other
aspects of this embodiment, a clostripain comprises a polypeptide
having, e.g., at least one, two, three, four, five, six, seven,
eight, nine, 10, 20, 30, 40, 50, or 100 contiguous amino acid
additions relative to SEQ ID NO: 38.
[0211] Other examples of a di-chain loop protease include SEQ ID
NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43,
SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID
NO: 48, and SEQ ID NO: 49, or a naturally-occurring of
non-naturally occurring variant.
[0212] It is envisioned that any and all assay conditions suitable
for proteolytic cleavage of the scissile bond comprising a di-chain
protease cleavage site by a di-chain loop protease are useful in
the methods disclosed in the present specification, such as, e.g.,
linear assay conditions and non-linear assay conditions. In an
embodiment of the present invention, the assay conditions are
linear. In an aspect of this embodiment, the assay amount of a
recombinantly-expressed or a modified Clostridial toxin is in
excess. In an aspect of this embodiment, the assay amount of a
di-chain loop protease is in excess. In another aspect of this
embodiment, the assay amount of a recombinantly-expressed or a
modified Clostridial toxin is rate-limiting. In another aspect of
this embodiment, the assay amount of a di-chain loop protease is
rate-limiting.
[0213] In other aspects of this embodiment, assay conditions
suitable for activating a recombinantly-expressed or modified
Clostridial toxin can be provided such that, e.g., at least 10% of
the recombinantly-expressed or modified Clostridial toxin is
cleaved, at least 20% of the recombinantly-expressed or modified
Clostridial toxin is cleaved, at least 30% of the
recombinantly-expressed or modified Clostridial toxin is cleaved,
at least 40% of the recombinantly-expressed or modified Clostridial
toxin is cleaved, at least 50% of the recombinantly-expressed or
modified Clostridial toxin is cleaved, at least 60% of the
recombinantly-expressed or modified Clostridial toxin is cleaved,
at least 70% of the recombinantly-expressed or modified Clostridial
toxin is cleaved, at least 80% of the recombinantly-expressed or
modified Clostridial toxin is cleaved or at least 90% of the
recombinantly-expressed or modified Clostridial toxin is cleaved.
In other aspects of this embodiment, conditions suitable for
activating a recombinantly-expressed or a modified Clostridial
toxin can be provided such that, e.g., at most 10% of the
recombinantly-expressed or modified Clostridial toxin is cleaved,
at most 20% of the recombinantly-expressed or modified Clostridial
toxin is cleaved, at most 30% of the recombinantly-expressed or
modified Clostridial toxin is cleaved, at most 40% of the
recombinantly-expressed or modified Clostridial toxin is cleaved,
at most 50% of the recombinantly-expressed or modified Clostridial
toxin is cleaved, at most 60% of the recombinantly-expressed or
modified Clostridial toxin is cleaved, at most 70% of the
recombinantly-expressed or modified Clostridial toxin is cleaved,
at most 80% of the recombinantly-expressed or modified Clostridial
toxin is cleaved or at most 90% of the recombinantly-expressed or
modified Clostridial toxin is cleaved. In another aspect of this
embodiment, conditions suitable for activating a
recombinantly-expressed or a modified Clostridial toxin can be
provided such that 100% of the recombinantly-expressed or modified
Clostridial toxin is cleaved. In another aspect of this embodiment,
the conditions suitable for activating a recombinantly-expressed or
a modified Clostridial toxin are provided such that the assay is
linear. In another aspect of this embodiment, the conditions
suitable for activating a recombinantly-expressed or a modified
Clostridial toxin are provided such that the assay is
non-linear.
[0214] The presence of calcium ions is essential for Clostripain
proteolytic activity. Thus, in another embodiment, assay conditions
suitable for activating a recombinantly-expressed or modified
Clostridial toxin include a source of calcium, such as calcium
chloride or calcium acetate. In aspects of this embodiment, assay
conditions include calcium in the range of about 0.1 .mu.M to about
500 .mu.M, for example, about 0.1 .mu.M to about 50 .mu.M, about
0.1 .mu.M to about 5 .mu.M, about 1 .mu.M to about 500 .mu.M, about
1 .mu.M to about 50 .mu.M, about 1 .mu.M to about 5 .mu.M, about 5
.mu.M to about 15 .mu.M, and about 5 .mu.M to about 10 .mu.M. One
skilled in the art understands that calcium chelators such as EGTA
generally are excluded from an assay condition used to activate a
recombinantly-expressed or modified Clostridial toxin. Potent
inhibitors of clostripain activity include, e.g., oxidizing agents,
thiol-blocking agents, Co.sup.2+, Cu.sup.2+, Cd.sup.2+ and heavy
metal ions. Citrate, borate and Tris partially inhibit Clostripain
proteolytic activity.
[0215] In addition, the activity of clostripain depends upon a
cysteine thiol group, so a reducing agent such as, e.g.,
dithiothreitol (DTT), cysteine, .beta.-mercaptoethanol,
dimethylsulfoxide (DMSO), or other sulfhydryl containing reagents
is included in the assay buffer. In aspect of this embodiment,
concentrations for a reducing agent may include, e.g., at least 10
nM, at least 50 nM, at least 100 nM, at least 500 nM, at least 1
mM, at least 10 mM or at least 100 mM. In another aspect of this
embodiment, concentrations for a reducing agent may include, e.g.,
at most 10 nM, at most 50 nM, at most 100 nM, at most 500 nM, at
most 1 mM, at most 10 mM or at most 100 mM. Non-limiting examples
of how to make and use specific reducing agents are described in,
e.g., MOLECULAR CLONING, A LABORATORY MANUAL, supra, (2001); and
CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004).
[0216] In another embodiment, the amount of di-chain loop protease
used to activate a recombinantly-expressed or a modified
Clostridial toxin can be in the range of about 0.001 .mu.g to about
500 .mu.g, for example, from about 0.001 .mu.g to about 05 .mu.g,
about 0.001 .mu.g to about 5 .mu.g, about 0.001 .mu.g to about 50
.mu.g, about 0.01 .mu.g to about 05 .mu.g, 0.01 .mu.g to about 5
.mu.g, about 0.01 .mu.g to about 50 .mu.g, about 0.01 .mu.g to
about 500 .mu.g, about 0.1 .mu.g to about 05 .mu.g, 0.1 .mu.g to
about 5 .mu.g, about 0.1 .mu.g to about 50 .mu.g, about 0.1 .mu.g
to about 500 .mu.g, about 1 .mu.g to about 05 .mu.g, about 1 .mu.g
to about 5 .mu.g, about 1 .mu.g to about 50 .mu.g, or about 1 .mu.g
to about 500 .mu.g.
[0217] In another embodiment, the pH of the buffer used in the
method to activate a recombinantly-expressed or a modified
Clostridial toxin can be in the range of about pH 6.0 to about pH
9.5, for example, from about pH 6.0 to about pH 9.0, about pH 6.0
to about pH 8.5, about pH 6.0 to about pH 8.0, about pH 6.0 to
about pH 7.5, about pH 7.0 to about pH 9.0, about pH 7.0 to about
pH 8.5, about pH 7.0 to about pH 8.0, about pH 7.2 to about pH 8.0,
about pH 7.2 to about pH 7.8, about pH 7.2 to about pH 7.6, about
pH 7.2 to about pH 7.4, about pH 7.4 to about pH 8.0, about pH 7.4
to about pH 7.8, about pH 7.4 to about pH 7.6, about pH 7.4 to
about pH 8.0, about pH 7.4 to about pH 7.8, or about pH 7.4 to
about pH 7.6.
[0218] In a further embodiment, it is also envisioned that any and
all buffers that allow the cleavage of the di-chain loop protease
cleavage site by a di-chain loop protease can optionally be used in
the activation methods disclosed in the present specification.
Assay buffers can be varied as appropriate by one skilled in the
art and generally depend, in part, on the pH value desired for the
assay and the detection means employed. Therefore, aspects of this
embodiment may optionally include, e.g.,
2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffers; Phosphate
buffers, such as, e.g., potassium phosphate buffers and sodium
phosphate buffers; Good buffers, such as, e.g., 2-(N-morpholino)
ethanesulfonic acid (MES), piperazine-N,N'-bis(2-ethanesulfonic
acid)(PIPES), N,N'-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid
(BES), 3-(N-morpholino) propanesulfonic acid (MOPS),
N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES),
piperazine-N,N'-bis(2-hydroxypropanesulfonic acid) (POPSO),
N-tris(hydroxymethyl)methylglycine (Tricine),
N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),
3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic
acid (AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid
(CAPSO), and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS);
saline buffers, such as, e.g., Phosphate-buffered saline (PBS),
HEPES-buffered saline, and Tris-buffered saline (TBS); Acetate
buffers, such as, e.g., magnesium acetate, potassium actetate, and
Tris acetate; and the like, or any combination thereof. In
addition, the buffer concentration in a method disclosed in the
present specification can be varied as appropriate by one skilled
in the art and generally depend, in part, on the buffering capacity
of a particular buffer being used and the detection means employed.
Thus, aspects of this embodiment may include a buffer concentration
of, e.g., at least 1 mM, at least 5 mM, at least 10 mM, at least 20
mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM,
at least 70 mM, at least 80 mM, at least 90 mM, or at least 100 mM.
Non-limiting examples of how to make and use specific buffers are
described in, e.g., MOLECULAR CLONING, A LABORATORY MANUAL, supra,
(2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra,
(2004).
[0219] In a further embodiment, it is also envisioned that any and
all salts that allow the cleavage of the di-chain loop protease
cleavage site by a di-chain loop protease can optionally be used in
the activation methods disclosed in the present specification.
Assay salts can be varied as appropriate by one skilled in the art
and generally depend, in part, on the physiological conditions
desired for the assay and the detection means employed. Therefore,
aspects of this embodiment may optionally include, e.g., sodium
chloride, potassium chloride, calcium chloride, magnesium chloride,
manganese chloride, zinc chloride, magnesium sulfate, zinc sulfate,
and the like, or any combination thereof. In addition, the salt
concentration in a method disclosed in the present specification
can be varied as appropriate by one skilled in the art and
generally depend, in part, on the buffering capacity of a
particular buffer being used and the detection means employed.
Thus, aspects of this embodiment may include a salt concentration
of, e.g., at least 1 mM, at least 5 mM, at least 10 mM, at least 20
mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM,
at least 70 mM, at least 80 mM, at least 90 mM, or at least 100 mM.
Non-limiting examples of how to make and use specific salts are
described in, e.g., MOLECULAR CLONING, A LABORATORY MANUAL, supra,
(2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra,
(2004).
[0220] In another embodiment, the concentration of a
recombinantly-expressed or a modified Clostridial toxin to be
activated can be in the range of about 0.0001 ng/ml to 500 .mu.g/ml
toxin, for example, about 0.0001 ng/ml to 50 .mu.g/ml toxin, 0.001
ng/ml to 500 .mu.g/ml toxin, 0.001 ng/ml to 50 .mu.g/ml toxin,
0.0001 to 5000 ng/ml toxin, 0.001 ng/ml to 5000 ng/ml, 0.01 ng/ml
to 5000 ng/ml, 0.1 ng/ml to 5000 ng/ml, 0.1 ng/ml to 500 ng/ml, 0.1
ng/ml to 50 ng/ml, 1 ng/ml to 5000 ng/ml, 1 ng/ml to 500 ng/ml, 1
ng/ml to 50 ng/ml, 10 ng/ml to 5000 ng/ml, 10 ng/ml to 500 ng/ml,
50 ng/ml to 5000 ng/ml, 50 ng/ml to 500 ng/ml or 100 ng/ml to 5000
ng/ml toxin. In another embodiment, the concentration of a
recombinantly-expressed or a modified Clostridial toxin to be
activated can be in the range of about 0.1 pM to 500 .mu.M, 0.1 pM
to 100 .mu.M, 0.1 pM to 10 .mu.M, 0.1 pM to 1 .mu.M, 0.1 pM to 500
nM, 0.1 pM to 100 nM, 0.1 pM to 10 nM, 0.1 pM to 1 nM, 0.1 pM to
500 pM, 0.1 pM to 100 pM, 0.1 pM to 50 pM, 0.1 pM to 10 pM, 1 pM to
500 .mu.M, 1 pM to 100 .mu.M, 1 pM to 10 .mu.M, 1 pM to 1 .mu.M, 1
pM to 500 nM, 1 pM to 100 nM, 1 pM to 10 nM, 1 pM to 1 nM, 1 pM to
500 pM, 1 pM to 100 pM, 1 pM to 50 pM, 1 pM to 10 pM, 10 pM to 500
.mu.M, 10 pM to 100 .mu.M, 10 pM to 10 .mu.M, 10 pM to 10 .mu.M, 10
pM to 500 nM, 10 pM to 100 nM, 10 pM to 10 nM, 10 pM to 1 nM, 10 pM
to 500 pM, 10 pM to 100 pM, 10 pM to 50 pM, 100 pM to 500 .mu.M,
100 pM to 100 .mu.M, 100 pM to 10 .mu.M, 100 pM to 1 .mu.M, 100 pM
to 500 nM, 100 pM to 100 nM, 100 pM to 10 nM, 100 pM to 1 nM, 100
pM to 500 pM 1 nM to 500 .mu.M, 1 nM to 100 .mu.M, 1 nM to 10
.mu.M, 1 nM to 1 .mu.M, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50
nM, 1 nM to 10 nM, 3 nM to 100 nM toxin. One skilled in the art
understands that the concentration of a recombinantly-expressed or
a modified Clostridial toxin to be activated will depend on the
particular recombinantly-expressed or modified Clostridial toxin to
be activated, as well as, the particular di-chain loop protease
used, the presence of inhibitory components, and the assay
conditions.
[0221] In still another embodiment, it is envisioned that any and
all temperatures that allow activation of a recombinantly-expressed
or a modified Clostridial toxin by a di-chain loop protease can be
used in methods disclosed in the present specification. Assay
temperatures can be varied as appropriate by one skilled in the art
and generally depend, in part, on the concentration, purity of the
recombinantly-expressed or modified Clostridial toxin, the activity
of the di-chain loop protease, the assay time or the convenience of
the artisan. Thus, an assay temperature should not be as low as to
cause the solution to freeze and should not be as high as to
denature a recombinantly-expressed or a modified Clostridial toxin
or a di-chain loop protease disclosed in the present specification.
In an aspect of this embodiment, the activation method is performed
within a temperature range above 0.degree. C., but below 40.degree.
C. In another aspect of this embodiment, the activation method is
performed within a temperature range of about 4.degree. C. to about
37.degree. C. In yet another aspect of this embodiment, the
activation method is performed within a temperature range of about
2.degree. C. to 10.degree. C. In yet another aspect of this
embodiment, the activation method is performed at about 4.degree.
C. In still another aspect of this embodiment, the activation
method is performed within a temperature range of about 10.degree.
C. to about 18.degree. C. In still another aspect of this
embodiment, the activation method is performed at about 16.degree.
C. In yet another aspect of this embodiment, the activation method
is performed within a temperature range of about 18.degree. C. to
about 32.degree. C. In yet another aspect of this embodiment, the
activation method is performed at about 20.degree. C. In another
aspect of this embodiment, the activation method is performed
within a temperature range of about 32.degree. C. to about
40.degree. C. In another aspect of this embodiment, the activation
method is performed at about 37.degree. C.
[0222] In still another embodiment, it is envisioned that any and
all times sufficient for activating a recombinantly-expressed or a
modified Clostridial toxin can be used in methods disclosed in the
present specification. Assay times can be varied as appropriate by
the skilled artisan and generally depend, in part, on the
concentration and purity of the recombinantly-expressed or a
modified Clostridial toxin, activity of the di-chain loop protease,
incubation temperature or the convenience of the artisan. Assay
times generally vary, without limitation, in the range of about 15
minutes to about 4 hours, 30 minutes to 8 hours, 1 hour to 12
hours, 2 hours to 24 hours, 4 hours to 48 hours, 6 hours to 72
hours. It is understood that assays can be terminated at any
time.
[0223] Aspects of the present invention can also be described as
follows: [0224] 1. A modified Clostridial toxin comprising an
exogenous Clostridial toxin di-chain loop region including a
di-chain protease cleavage site; wherein the Clostridial toxin
di-chain loop region replaces an endogenous Clostridial toxin
di-chain loop region. [0225] 2. The modified Clostridial toxin of
1, wherein the Clostridial toxin being modified is BoNT/B,
[0226] BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, TeNT, BaNT, or BuNT
and the exogenous Clostridial toxin di-chain loop region is BoNT/A.
[0227] 3. The modified Clostridial toxin of 2, wherein the modified
Clostridial toxin is SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52,
SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID
NO: 57, or SEQ ID NO: 58. [0228] 4. The modified Clostridial toxin
of 1, wherein the Clostridial toxin being modified is BoNT/A,
BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, TeNT, BaNT, or BuNT and
the exogenous Clostridial toxin di-chain loop region is BoNT/B.
[0229] 5. The modified Clostridial toxin of 1, wherein the
Clostridial toxin being modified is BoNT/A, BoNT/B, BoNT/C1,
BoNT/D, BoNT/E, BoNT/G, TeNT, BaNT, or BuNT and the exogenous
Clostridial toxin di-chain loop region is BoNT/F. [0230] 6. The
modified Clostridial toxin of 1, wherein the Clostridial toxin
being modified is BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/G,
BaNT, or BuNT and the exogenous Clostridial toxin di-chain loop
region is TeNT. [0231] 7. The modified Clostridial toxin of 1,
wherein the Clostridial toxin being modified is BoNT/A, BoNT/B,
BoNT/C1, BoNT/D, BoNT/E, BoNT/G, TeNT, or BuNT and the exogenous
Clostridial toxin di-chain loop region is BaNT. [0232] 8. A
polynucleotide molecule encoding a modified Clostridial toxin
according to any one of 1-7. [0233] 9. The polynucleotide molecule
of 8, further comprising an expression vector. [0234] 10. A method
of producing a modified Clostridial toxin comprising the step of
expressing in a cell a polynucleotide molecule according to 9,
wherein expression from the polynucleotide molecule produces the
encoded modified Clostridial toxin. [0235] 11. A method of
producing a modified Clostridial toxin comprising the steps of:
[0236] a. introducing into a cell a polynucleotide molecule as
defined in 9; and [0237] b. expressing the polynucleotide molecule,
wherein expression from the polynucleotide molecule produces the
encoded modified Clostridial toxin. [0238] 12. A method of
activating a modified Clostridial toxin, the method comprising the
step of incubating a modified Clostridial toxin according to any
one of 1-7 with a di-chain loop protease, wherein cleavage of the
modified Clostridial toxin by the di-chain loop protease converts
the modified Clostridial toxin from its single-chain polypeptide
form into its di-chain form, thereby activating the modified
Clostridial toxin. [0239] 13. A method of activating a modified
Clostridial toxin, the method comprising the step of incubating a
modified Clostridial toxin according to any one of 2 with a BoNT/A
di-chain loop protease under physiological conditions; [0240]
wherein cleavage of the modified Clostridial toxin by the BoNT/A
di-chain loop protease converts the modified Clostridial toxin from
its single-chain polypeptide form into its di-chain form, thereby
activating the modified Clostridial toxin. [0241] 14. A method of
either 13 Or 14, wherein the BoNT/A toxin di-chain loop protease is
SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID
NO: 37, or SEQ ID NO: 38; and [0242] 15. A method of activating a
recombinatly-expressed Clostridial toxin, the method comprising the
steps of: [0243] a. expressing in an aerobic bacterial cell a
polynucleotide molecule encoding a Clostridial toxin; [0244] b.
purifying the Clostridial toxin; and [0245] c. incubating the
purified Clostridial toxin with a Clostridial toxin di-chain loop
protease under physiological conditions; [0246] wherein cleavage of
the purified Clostridial toxin by the Clostridial toxin di-chain
loop protease converts the purified Clostridial toxin from its
single-chain polypeptide form into its di-chain form, thereby
activating the recombinatly-expressed Clostridial toxin. [0247] 16.
A method of activating a recombinatly-expressed BoNT/A, the method
comprising the steps of: [0248] a. expressing in an aerobic
bacterial cell a polynucleotide molecule encoding a BoNT/A; [0249]
b. purifying the BoNT/A; and [0250] c. incubating the purified
BoNT/A with a BoNT/A di-chain loop protease under physiological
conditions; [0251] wherein the BoNT/A toxin di-chain loop protease
is SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ
ID NO: 37, or SEQ ID NO: 38; and [0252] wherein cleavage of the
purified BoNT/A by the BoNT/A di-chain loop protease converts the
purified BoNT/A from its single-chain polypeptide form into its
di-chain form, thereby activating the recombinatly-expressed
BoNT/A. [0253] 17. A modified Clostridial toxin comprising: [0254]
a) a CLostridial toxin enzymatic domain; [0255] b) a Clostridial
toxin translocation domain; [0256] c) a targeting moiety; [0257] d)
an exogenous Clostridial toxin di-chain loop region including a
di-chain protease cleavage site; wherein the Clostridial toxin
di-chain loop region replaces an endogenous Clostridial toxin
di-chain loop region. [0258] 18. The modified Clostridial toxin of
17, wherein the targeting moiety is one disclosed in Steward,
supra, International Patent Publication No. 2006/008956; Steward,
supra, U.S. patent application Ser. No. 11/776,043; Steward, supra,
International Patent Publication No. 2006/009831; Steward, supra,
U.S. Patent Publication No. 2006/0211619; Steward, supra, U.S.
patent application Ser. No. 11/776,052; Foster, supra, U.S. Pat.
No. 5,989,545; Shone, supra, U.S. Pat. No. 6,461,617; Quinn, supra,
U.S. Pat. No. 6,632,440; Steward, supra, U.S. Pat. No. 6,843,998;
Donovan, supra, U.S. Pat. No. 7,138,127; Foster, supra, U.S. Patent
Publication 2003/0180289; Dolly, supra, U.S. Pat. No. 7,132,259;
Foster, supra, International Patent Publication WO 2005/023309;
Steward, supra, U.S. patent application Ser. No. 11/376,696;
Foster, supra, International Patent Publication WO 2006/059093;
Foster, supra, International Patent Publication WO 2006/059105; or
Steward, supra, U.S. patent application Ser. No. 11/776,075.
EXAMPLES
[0259] The following non-limiting examples are provided for
illustrative purposes only in order to facilitate a more complete
understanding of disclosed embodiments and are in no way intended
to limit any of the embodiments disclosed in the present
specification.
Example 1
Construction of Modified Clostridial Toxins Comprising a Di-Chain
Loop Protease Cleavage Site from a Different Clostridial Toxin
[0260] This example illustrates how to make a modified Clostridial
toxin comprising a di-chain loop protease cleavage site from a
different Clostridial toxin located in the di-chain loop region of
the modified toxin.
[0261] A polynucleotide molecule based on BoNT/E-DiA (SEQ ID NO:
50) will be synthesized using standard procedures (BlueHeron.RTM.
Biotechnology, Bothell, Wash.). BoNT/E-DiA is a toxin which is
modified to replace the endogenous di-chain loop region of BoNT/E
(SEQ ID NO: 15) with the BoNT/A di-chain loop region of SEQ ID NO:
11. Oligonucleotides of 20 to 50 bases in length are synthesized
using standard phosphoramidite synthesis. These oligonucleotides
are hybridized into double stranded duplexes that are ligated
together to assemble the full-length polynucleotide molecule. This
polynucleotide molecule is cloned using standard molecular biology
methods into a pUCBHB1 vector at the SmaI site to generate
pUCBHB1/BoNT/E-DiA. The synthesized polynucleotide molecule is
verified by sequencing using Big Dye Terminator.TM. Chemistry 3.1
(Applied Biosystems, Foster City, Calif.) and an ABI 3100 sequencer
(Applied Biosystems, Foster City, Calif.).
[0262] If desired, an expression optimized polynucleotide molecule
based on BoNT/E-DiA (SEQ ID NO: 50) can be synthesized in order to
improve expression in an Escherichia coli strain. The
polynucleotide molecule encoding the BoNT/E-DiA can be modified to
1) contain synonymous codons typically present in native
polynucleotide molecules of an Escherichia coli strain; 2) contain
a G+C content that more closely matches the average G+C content of
native polynucleotide molecules found in an Escherichia coli
strain; 3) reduce polymononucleotide regions found within the
polynucleotide molecule; and/or 4) eliminate internal regulatory or
structural sites found within the polynucleotide molecule, see,
e.g., Lance E. Steward et al. Optimizing Expression of Active
Botulinum Toxin Type E, International Patent Publication No. WO
2006/011966 (Feb. 2, 2006); Lance E. Steward et al. Optimizing
Expression of Active Botulinum Toxin Type A, International Patent
Publication No. WO 2006/017749 (Feb. 16, 2006), each of which is
hereby incorporated by reference in its entirety. Once sequence
optimization is complete, oligonucleotides of 20 to 50 bases in
length are synthesized using standard phosphoramidite synthesis.
These oligonucleotides are hybridized into double stranded duplexes
that are ligated together to assemble the full-length
polynucleotide molecule. This polynucleotide molecule is cloned
using standard molecular biology methods into a pUCBHB1 vector at
the SmaI site to generate pUCBHB1/BoNT/E-DiA. The synthesized
polynucleotide molecule is verified by sequencing using Big Dye
Terminator.TM. Chemistry 3.1 (Applied Biosystems, Foster City,
Calif.) and an ABI 3100 sequencer (Applied Biosystems, Foster City,
Calif.). If so desired, optimization to a different organism, such
as, e.g., a yeast strain, an insect cell-line or a mammalian cell
line, can be done, see, e.g., Steward, supra, International Patent
Publication No. WO 2006/011966 (Feb. 2, 2006); and Steward, supra,
International Patent Publication No. WO 2006/017749 (Feb. 16,
2006).
[0263] A similar cloning strategy is used to make pUCBHB1 cloning
constructs for BoNT/E-DiB, a modified BoNT/E where SEQ ID NO: 15 is
replaced with SEQ ID NO: 12, a BoNT/B di-chain loop region;
BoNT/E-DiF, a modified BoNT/E where SEQ ID NO: 15 is replaced with
SEQ ID NO: 16, a BoNT/F di-chain loop region; BoNT/E-DiBa, a
modified BoNT/E where SEQ ID NO: 15 is replaced with SEQ ID NO: 19,
a BaNT di-chain loop region; and BoNT/E-DiT, a modified BoNT/E
where SEQ ID NO: 15 is replaced with SEQ ID NO: 18, a TeNT di-chain
loop region. In addition, using a similar strategy one skilled in
the art can, e.g., modify BoNT/B by replacing the BoNT/B di-chain
loop region of SEQ ID NO: 12 with SEQ ID NO: 11, a BoNT/A di-chain
loop region, to construct BoNT/B-Di-A (SEQ ID NO: 51); modify
BoNT/C1 by replacing the BoNT/C1 di-chain loop region of SEQ ID NO:
13 with SEQ ID NO: 11, a BoNT/A di-chain loop region, to construct
BoNT/C1-Di-A (SEQ ID NO: 52); modify BoNT/D by replacing the BoNT/D
di-chain loop region of SEQ ID NO: 14 with SEQ ID NO: 11, a BoNT/A
di-chain loop region, to construct BoNT/D-Di-A (SEQ ID NO: 53);
modify BoNT/F by replacing the BoNT/F di-chain loop region of SEQ
ID NO: 16 with SEQ ID NO: 11, a BoNT/A di-chain loop region, to
construct BoNT/F-Di-A (SEQ ID NO: 54); modify BoNT/G by replacing
the BoNT/G di-chain loop region of SEQ ID NO: 17 with SEQ ID NO:
11, a BoNT/A di-chain loop region, to construct BoNT/G-Di-A (SEQ ID
NO: 55); modify TeNT by replacing the TeNT di-chain loop region of
SEQ ID NO: 18 with SEQ ID NO: 11, a BoNT/A di-chain loop region, to
construct TeNT-Di-A (SEQ ID NO: 56); modify BaNT by replacing the
BaNT di-chain loop region of SEQ ID NO: 19 with SEQ ID NO: 11, a
BoNT/A di-chain loop region, to construct BaNT-Di-A (SEQ ID NO:
57); and modify BuNT by replacing the BuNT di-chain loop region of
SEQ ID NO: 20 with SEQ ID NO: 11, a BoNT/A di-chain loop region, to
construct BuNT-Di-A (SEQ ID NO: 58).
[0264] To construct pET29/BoNT/E-DiA, a pUCBHB1/BoNT/E-DiA
construct is digested with restriction endonucleases that 1) will
excise the polynucleotide molecule encoding BoNT/E-DiA; and 2) will
enable this polynucleotide molecule to be operably-linked to a
pET29 vector (EMD Biosciences-Novagen, Madison, Wis.). This insert
will be subcloned using a T4 DNA ligase procedure into a pET29
vector that is digested with appropriate restriction endonucleases
to yield pET29/BoNT/E-DiA. The ligation mixture will be transformed
into chemically competent E. coli DH5a cells (Invitrogen, Inc,
Carlsbad, Calif.) using a heat shock method, will be plated on 1.5%
Luria-Bertani agar plates (pH 7.0) containing 50 .mu.g/mL of
Kanamycin, and will be placed in a 37.degree. C. incubator for
overnight growth. Bacteria containing expression constructs will be
identified as Kanamycin resistant colonies. Candidate constructs
will be isolated using an alkaline lysis plasmid mini-preparation
procedure and will be analyzed by restriction endonuclease digest
mapping to determine the presence and orientation of the insert.
This cloning strategy will yield a pET29 expression construct
comprising the polynucleotide molecule encoding the BoNT/E-DiA
operably-linked to a carboxyl terminal polyhistidine affinity
binding peptide.
[0265] A similar cloning strategy can be used to make pET29
expression constructs comprising the polynucleotide molecule
encoding BoNT/E-DiB, BoNT/E-DiF, BoNT/E-Ba and BoNT/E-DiT.
Likewise, a similar cloning strategy will be used to make pET29
expression constructs comprising a polynucleotide molecule encoding
BoNT/B-DiA, BoNT/C1-DiA, BoNT/D-DiA, BoNT/F-DiA, BoNT/G-DiA,
TeNT-DiA, BaNT-DiA, and BuNT-DiA.
Example 2
Expression of Modified Clostridial Toxins in a Bacterial Cell
[0266] The following example illustrates a procedure useful for
expressing any of the modified Clostridial toxins disclosed in the
present specification in a bacterial cell.
[0267] An expression construct, such as, e.g., pET29/BoNT/E-DiA,
see, e.g., Example 1 is introduced into chemically competent E.
coli BL21 (DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a
heat-shock transformation protocol. The heat-shock reaction is
plated onto 1.5% Luria-Bertani agar plates (pH 7.0) containing 50
.mu.g/mL of Kanamycin and is placed in a 37.degree. C. incubator
for overnight growth. Kanamycin-resistant colonies of transformed
E. coli containing the expression construct, such as, e.g.,
pET29/BoNT/E-DiA are used to inoculate a baffled flask containing
3.0 mL of PA-0.5G media containing 50 .mu.g/mL of Kanamycin which
is then placed in a 37.degree. C. incubator, shaking at 250 rpm,
for overnight growth. The resulting overnight starter culture is in
turn used to inoculate a 3 L baffled flask containing ZYP-5052
autoinducing media containing 50 .mu.g/mL of Kanamycin at a
dilution of 1:1000. Culture volumes ranged from about 600 mL (20%
flask volume) to about 750 mL (25% flask volume). These cultures
are grown in a 37.degree. C. incubator shaking at 250 rpm for
approximately 5.5 hours and are then transferred to a 16.degree. C.
incubator shaking at 250 rpm for overnight expression. Cells are
harvested by centrifugation (4,000 rpm at 4.degree. C. for 20-30
minutes) and are used immediately, or stored dry at -80.degree. C.
until needed.
Example 3
Purification and Quantification of Modified Clostridial Toxins
[0268] The following example illustrates methods useful for
purification and quantification of any modified Clostridial toxins
disclosed in the present specification.
[0269] For immobilized metal affinity chromatography (IMAC) protein
purification, E. coli BL21 (DE3) cell pellets used to express a
modified Clostridial toxin, as described in Example 2, are
resuspended in Column Binding Buffer (25 mM N-(2-hydroxyethyl)
piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM
sodium chloride; 10 mM imidazole; 2.times. Protease Inhibitor
Cocktail Set III (EMD Biosciences-Calbiochem, San Diego Calif.); 5
units/mL of Benzonase (EMD Biosciences-Novagen, Madison, Wis.);
0.1% (v/v) Triton-X.RTM. 100, 4-octylphenol polyethoxylate; 10%
(v/v) glycerol), and then are transferred to a cold Oakridge
centrifuge tube. The cell suspension is sonicated on ice (10-12
pulses of 10 seconds at 40% amplitude with 60 seconds cooling
intervals on a Branson Digital Sonifier) in order to lyse the cells
and then is centrifuged (16,000 rpm at 4.degree. C. for 20 minutes)
to clarify the lysate. An immobilized metal affinity chromatography
column is prepared using a 20 mL Econo-Pac column support (Bio-Rad
Laboratories, Hercules, Calif.) packed with 2.5-5.0 mL of TALON.TM.
SuperFlow Co.sup.2+ affinity resin (BD Biosciences-Clontech, Palo
Alto, Calif.), which is then equilibrated by rinsing with 5 column
volumes of deionized, distilled water, followed by 5 column volumes
of Column Binding Buffer. The clarified lysate is applied slowly to
the equilibrated column by gravity flow (approximately 0.25-0.3
mL/minute). The column is then washed with 5 column volumes of
Column Wash Buffer (N-(2-hydroxyethyl)
piperazine-N'-(2-ethanesulfonic acid) (HEPES), pH 7.8; 500 mM
sodium chloride; 10 mM imidazole; 0.1% (v/v) Triton-X.RTM. 100,
4-octylphenol polyethoxylate; 10% (v/v) glycerol). The Clostridial
toxin is eluted with 20-30 mL of Column Elution Buffer (25 mM
N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES),
pH 7.8; 500 mM sodium chloride; 500 mM imidazole; 0.1% (v/v)
Triton-X.RTM. 100, 4-octylphenol polyethoxylate; 10% (v/v)
glycerol) and is collected in approximately twelve 1 mL fractions.
The amount of Clostridial toxin contained in each elution fraction
is determined by a Bradford dye assay. In this procedure, 20 .mu.L
aliquots of each 1.0 mL fraction is combined with 200 .mu.L of
Bio-Rad Protein Reagent (Bio-Rad Laboratories, Hercules, Calif.),
diluted 1 to 4 with deionized, distilled water, and then the
intensity of the colorimetric signal is measured using a
spectrophotometer. The five fractions with the strongest signal are
considered the elution peak and are combined together. Total
protein yield is determined by estimating the total protein
concentration of the pooled peak elution fractions using bovine
gamma globulin as a standard (Bio-Rad Laboratories, Hercules,
Calif.).
[0270] For purification of a modified Clostridial toxin using a
FPLC desalting column, a HiPrep.TM. 26/10 size exclusion column
(Amersham Biosciences, Piscataway, N.J.) is pre-equilibrated with
80 mL of 4.degree. C. Column Buffer (50 mM sodium phosphate, pH
6.5). After the column is equilibrated, a Clostridial toxin sample
is applied to the size exclusion column with an isocratic mobile
phase of 4.degree. C. Column Buffer and at a flow rate of 10
mL/minute using a BioLogic DuoFlow chromatography system (Bio-Rad
Laboratories, Hercules, Calif.). The desalted modified Clostridial
toxin sample is collected as a single fraction of approximately
7-12 mL.
[0271] For purification of a modified Clostridial toxin using a
FPLC ion exchange column, a Clostridial toxin sample that has been
desalted following elution from an IMAC column is applied to a 1 mL
Q1.TM. anion exchange column (Bio-Rad Laboratories, Hercules,
Calif.) using a BioLogic DuoFlow chromatography system (Bio-Rad
Laboratories, Hercules, Calif.). The sample is applied to the
column in 4.degree. C. Column Buffer (50 mM sodium phosphate, pH
6.5) and is eluted by linear gradient with 4.degree. C. Elution
Buffer (50 mM sodium phosphate, 1 M sodium chloride, pH 6.5) as
follows: step 1, 5.0 mL of 5% Elution Buffer at a flow rate of 1
mL/minute; step 2, 20.0 mL of 5-30% Elution Buffer at a flow rate
of 1 mL/minute; step 3, 2.0 mL of 50% Elution Buffer at a flow rate
of 1.0 mL/minute; step 4, 4.0 mL of 100% Elution Buffer at a flow
rate of 1.0 mL/minute; and step 5, 5.0 mL of 0% Elution Buffer at a
flow rate of 1.0 mL/minute. Elution of Clostridial toxin from the
column is monitored at 280, 260, and 214 nm, and peaks absorbing
above a minimum threshold (0.01 au) at 280 nm are collected. Most
of the Clostridial toxin will elute at a sodium chloride
concentration of approximately 100 to 200 mM. Average total yields
of Clostridial toxin will be determined by a Bradford assay.
[0272] Expression of a modified Clostridial toxin is analyzed by
polyacrylamide gel electrophoresis. Samples purified using the
procedure described above are added to 2.times.LDS Sample Buffer
(Invitrogen, Inc, Carlsbad, Calif.) and are separated by MOPS
polyacrylamide gel electrophoresis using NuPAGE.RTM. Novex 4-12%
Bis-Tris precast polyacrylamide gels (Invitrogen, Inc, Carlsbad,
Calif.) under denaturing, reducing conditions. Gels are stained
with SYPRO.RTM. Ruby (Bio-Rad Laboratories, Hercules, Calif.) and
the separated polypeptides are imaged using a Fluor-S MAX
Multilmager (Bio-Rad Laboratories, Hercules, Calif.) for
quantification of Clostridial toxin expression levels. The size and
amount of the Clostridial toxin is determined by comparison to
MagicMark.TM. protein molecular weight standards (Invitrogen, Inc,
Carlsbad, Calif.).
[0273] Expression of modified Clostridial toxin is also analyzed by
Western blot analysis. Protein samples purified using the procedure
described above are added to 2.times.LDS Sample Buffer (Invitrogen,
Inc, Carlsbad, Calif.) and are separated by MOPS polyacrylamide gel
electrophoresis using NuPAGE.RTM. Novex 4-12% Bis-Tris precast
polyacrylamide gels (Invitrogen, Inc, Carlsbad, Calif.) under
denaturing, reducing conditions. Separated polypeptides are
transferred from the gel onto polyvinylidene fluoride (PVDF)
membranes (Invitrogen, Inc, Carlsbad, Calif.) by Western blotting
using a Trans-Blot.RTM. SD semi-dry electrophoretic transfer cell
apparatus (Bio-Rad Laboratories, Hercules, Calif.). PVDF membranes
are blocked by incubating at room temperature for 2 hours in a
solution containing 25 mM Tris-Buffered Saline (25 mM
2-amino-2-hydroxymethyl-1,3-propanediol hydrochloric acid
(Tris-HCl)(pH 7.4), 137 mM sodium chloride, 2.7 mM potassium
chloride), 0.1% TWEEN-20.RTM., polyoxyethylene (20) sorbitan
monolaureate, 2% bovine serum albumin, 5% nonfat dry milk. Blocked
membranes are incubated at 4.degree. C. for overnight in
Tris-Buffered Saline TWEEN-20.RTM. (25 mM Tris-Buffered Saline,
0.1% TWEEN-20.RTM., polyoxyethylene (20) sorbitan monolaureate)
containing appropriate primary antibodies as a probe. Primary
antibody probed blots are washed three times for 15 minutes each
time in Tris-Buffered Saline TWEEN-20.RTM.. Washed membranes are
incubated at room temperature for 2 hours in Tris-Buffered Saline
TWEEN-20.RTM. containing an appropriate immunoglobulin G antibody
conjugated to horseradish peroxidase as a secondary antibody.
Secondary antibody-probed blots are washed three times for 15
minutes each time in Tris-Buffered Saline TWEEN-20.RTM.. Signal
detection of the labeled Clostridial toxin are visualized using the
ECL Plus.TM. Western Blot Detection System (Amersham Biosciences,
Piscataway, N.J.) and are imaged with a Typhoon 9410 Variable Mode
Imager (Amersham Biosciences, Piscataway, N.J.) for quantification
of modified Clostridial toxin expression levels.
Example 4
Expression of Modified Clostridial Toxins in a Yeast Cell
[0274] The following example illustrates a procedure useful for
expressing any of the modified Clostridial toxins disclosed in the
present specification in a yeast cell.
[0275] To construct a suitable yeast expression construct encoding
a modified Clostridial toxin, restriction endonuclease sites
suitable for cloning an operably linked polynucleotide molecule
into a pPIC A vector (Invitrogen, Inc, Carlsbad, Calif.) are
incorporated into the 5'- and 3' ends of the polynucleotide
molecule encoding BoNT/E-DiA of SEQ ID NO: 50. This polynucleotide
molecule is synthesized and a pUCBHB1/BoNT/E-DiA construct is
obtained as described in Example 1. This construct is digested with
restriction enzymes that 1) will excise the insert containing the
open reading frame encoding BoNT/E-DiA; and 2) enable this insert
to be operably-linked to a pPIC A vector. This insert is subcloned
using a T4 DNA ligase procedure into a pPIC A vector that is
digested with appropriate restriction endonucleases to yield pPIC
A/BoNT/E-DiA. The ligation mixture is transformed into chemically
competent E. coli DH5a cells (Invitrogen, Inc, Carlsbad, Calif.)
using a heat shock method, plated on 1.5% low salt Luria-Bertani
agar plates (pH 7.5) containing 25 .mu.g/mL of Zeocin.TM., and
placed in a 37.degree. C. incubator for overnight growth. Bacteria
containing expression constructs are identified as Zeocin.TM.
resistant colonies. Candidate constructs are isolated using an
alkaline lysis plasmid mini-preparation procedure and analyzed by
restriction endonuclease digest mapping to determine the presence
and orientation of the insert. This cloning strategy yielded a pPIC
A expression construct comprising the polynucleotide molecule
encoding the BoNT/E-DiA of SEQ ID NO: 50 operably-linked to a
carboxyl-terminal c-myc and polyhistidine binding peptides.
[0276] A similar cloning strategy is used to make pPIC A expression
constructs encoding BoNT/E-DiB, BoNT/E-DiF, BoNT/E-Ba, BoNT/E-DiT,
BoNT/B-DiA, BoNT/C1-DiA, BoNT/D-DiA, BoNT/F-DiA, BoNT/G-DiA,
TeNT-DiA, BaNT-DiA, and BuNT-DiA.
[0277] To construct a yeast cell line expressing a modified
Clostridial toxin, pPICZ A/BoNT/E-DiA is digested with a suitable
restriction endonuclease (i.e., SacI, PmeI or BstXI) and the
resulting linearized expression construct is transformed into an
appropriate P. pastoris Mut.sup.S strain KM71H using an
electroporation method. The transformation mixture is plated on
1.5% YPDS agar plates (pH 7.5) containing 100 .mu.g/mL of
Zeocin.TM. and placed in a 28-30.degree. C. incubator for 1-3 days
of growth. Selection of transformants integrating the pPICZ
A/BoNT/E-DiA at the 5' AOX1 locus is determined by colony
resistance to Zeocin.TM.. Cell lines integrating a pPICZ
A/BoNT/E-DiA construct is tested for BoNT/E-DiA expression using a
small-scale expression test. Isolated colonies from test cell lines
that have integrated pPICZ A/BoNT/E-DiA are used to inoculate 1.0 L
baffled flasks containing 100 mL of MGYH media and grown at about
28-30.degree. C. in a shaker incubator (250 rpm) until the culture
reaches an OD.sub.600=2-6 (approximately 16-18 hours). Cells are
harvested by centrifugation (3,000.times.g at 22.degree. C. for 5
minutes). To induce expression, the cell pellet is resuspended in
15 mL of MMH media and 100% methanol is added to a final
concentration of 0.5%. Cultures are grown at about 28-30.degree. C.
in a shaker incubator (250 rpm) for six days. Additional 100%
methanol is added to the culture every 24 hours to a final
concentration of 0.5%. A 1.0 mL test aliquot is taken from the
culture every 24 hours starting at time zero and ending at time 144
hours. Cells are harvested from the aliquots by microcentrifugation
to pellet the cells and lysed using three freeze-thaw rounds
consisting of -80.degree. C. for 5 minutes, then 37.degree. C. for
5 minutes. Lysis samples are added to 2.times.LDS Sample Buffer
(Invitrogen, Inc, Carlsbad, Calif.) and expression from established
cell lines is measured by Western blot analysis (as described in
Example 8) using either anti-BoNT/E, anti-myc or anti-His
antibodies in order to identify lines expressing BoNT/E-DiA. The P.
pastoris Mut.sup.S KM71H cell line showing the highest expression
level of BoNT/E-DiA is selected for large-scale expression using
commercial fermentation procedures. Procedures for large-scale
expression are as outlined above except the culture volume is
approximately 2.5 L MGYH media grown in a 5 L BioFlo 3000 fermentor
and concentrations of all reagents will be proportionally increased
for this volume. A similar procedure can be used to express a pPICZ
A construct encoding BoNT/E-DiB, BoNT/E-DiF, BoNT/E-Ba, BoNT/E-DiT,
BoNT/B-DiA, BoNT/C1-DiA, BoNT/D-DiA, BoNT/F-DiA, BoNT/G-DiA,
TeNT-DiA, BaNT-DiA, and BuNT-DiA.
[0278] BoNT/E-DiA is purified using the IMAC procedure, as
described in Example 3. Expression from each culture is evaluated
by a Bradford dye assay, polyacrylamide gel electrophoresis and
Western blot analysis (as described in Example 3) in order to
determine the amounts of BoNT/E-DiA produced.
Example 5
Expression of Modified Clostridial Toxins in an Insect Cell
[0279] The following example illustrates a procedure useful for
expressing any of the modified Clostridial toxins disclosed in the
present specification in an insect cell.
[0280] To construct suitable an insect expression construct
encoding a modified Clostridial toxin, restriction endonuclease
sites suitable for cloning an operably linked polynucleotide
molecule into a pBACgus3 vector (EMD Biosciences-Novagen, Madison,
Wis.) are incorporated into the 5'- and 3' ends of the
polynucleotide molecule encoding BoNT/E-DiA of SEQ ID NO: 50. This
polynucleotide molecule is synthesized and a pUCBHB1/BoNT/E-DiA
construct is obtained as described in Example 1. This construct is
digested with restriction enzymes that 1) will excise the insert
containing the open reading frame encoding BoNT/E-DiA; and 2)
enable this insert to be operably-linked to a pBACgus3 vector. This
insert is subcloned using a T4 DNA ligase procedure into a pBACgus3
vector that is digested with appropriate restriction endonucleases
to yield pBACgus3/BoNT/E-DiA. The ligation mixture is transformed
into chemically competent E. coli DH5a cells (Invitrogen, Inc,
Carlsbad, Calif.) using a heat shock method, plated on 1.5%
Luria-Bertani agar plates (pH 7.0) containing 100 .mu.g/mL of
Ampicillin, and placed in a 37.degree. C. incubator for overnight
growth. Bacteria containing expression constructs are identified as
Ampicillin resistant colonies. Candidate constructs are isolated
using an alkaline lysis plasmid mini-preparation procedure and
analyzed by restriction endonuclease digest mapping to determine
the presence and orientation of the insert. This cloning strategy
yielded a pBACgus3 expression construct comprising the
polynucleotide molecule encoding the BoNT/E-DiA of SEQ ID NO: 50
operably linked to an amino-terminal gp64 signal peptide and a
carboxyl-terminal, Thrombin cleavable, polyhistidine affinity
binding peptide.
[0281] A similar cloning strategy is used to make pBACgus3
expression constructs encoding BoNT/E-DiB, BoNT/E-DiF, BoNT/E-Ba,
BoNT/E-DiT, BoNT/B-DiA, BoNT/C1-DiA, BoNT/D-DiA, BoNT/F-DiA,
BoNT/G-DiA, TeNT-DiA, BaNT-DiA, and BuNT-DiA.
[0282] To express a modified Clostridial toxin using a baculoviral
expression system, about 2.5.times.10.sup.6 Sf9 cells are plated in
four 60 mm culture dishes containing 2 mL of BacVector.RTM. Insect
media (EMD Biosciences-Novagen, Madison, Wis.) and incubated for
approximately 20 minutes in a 28.degree. C. incubator. For each
transfection, a 50 .mu.L transfection solution is prepared in a 6
mL polystyrene tube by adding 25 .mu.L of BacVector.RTM. Insect
media containing 100 ng of a pBACgus3 construct encoding a modified
Clostridial toxin, such as, e.g., pBACgus3/BoNT/E-DiA, and 500 ng
TIowE transfer plasmid to 25 .mu.L of diluted Insect GeneJuice.RTM.
containing 5 .mu.L Insect GeneJuice.RTM. (EMD Biosciences-Novagen,
Madison, Wis.) and 20 .mu.L nuclease-free water and this solution
is incubated for approximately 15 minutes. After the 15 minute
incubation, add 450 .mu.L BacVector.RTM. media to the transfection
solution and mix gently. Using this stock transfection solution as
the 1/10 dilution make additional transfection solutions of 1/50,
1/250 and 1/1250 dilutions. Add 100 .mu.L of a transfection
solution to the Sf9 cells from one of the four 60 mm culture
dishes, twice washed with antibiotic-free, serum-free
BacVector.RTM. Insect media and incubate at 22.degree. C. After one
hour, add 6 mL of 1% BacPlaque agarose-BacVector.RTM. Insect media
containing 5% bovine serum albumin. After the agarose is
solidified, add 2 mL BacVector.RTM. Insect media containing 5%
bovine serum albumin to the transfected cells and transfer the
cells to a 28.degree. C. incubator for 3-5 days until plaques are
visible. After 3-5 days post-transfection, plaques in the monolayer
will be stained for .beta.-glucuronidase reporter gene activity to
test for the presence of recombinant virus plaques containing
pBACgus3/BoNT/E-DiA by incubating the washed monolayer with 2 mL of
BacVector.RTM. Insect media containing 30 .mu.L of 20 mg/mL X-Gluc
Solution (EMD Biosciences-Novagen, Madison, Wis.) for approximately
2 hours in a 28.degree. C. incubator.
[0283] After identifying candidate recombinant virus plaques,
several candidate virus plaques are eluted and plaque purified. To
elute a recombinant virus, transfer a plug containing a recombinant
virus plaque with a sterile Pasteur pipet to 1 mL BacVector.RTM.
Insect media (EMD Biosciences-Novagen, Madison, Wis.) in a sterile
screw-cap vial. Incubate the vial for approximately 2 hours at
22.degree. C. or for approximately 16 hours at 4.degree. C. For
each recombinant virus plaque, 2.5.times.10.sup.5 Sf9 cells are
plated in 35 mm culture dishes containing 2 mL of BacVector.RTM.
Insect media (EMD Biosciences-Novagen, Madison, Wis.) and incubated
for approximately 20 minutes in a 28.degree. C. incubator. Remove
the media and add 200 .mu.L of eluted recombinant virus. After one
hour, add 2 mL of 1% BacPlaque agarose-BacVector.RTM. Insect media
containing 5% bovine serum albumin. After the agarose is
solidified, add 1 mL BacVector.RTM. Insect media containing 5%
bovine serum albumin to the transfected cells and transfer the
cells to a 28.degree. C. incubator for 3-5 days until plaques are
visible. After 3-5 days post-transfection, plaques in the monolayer
will be stained for .beta.-glucuronidase reporter gene activity to
test for the presence of recombinant virus plaques containing
pBACgus3/BoNT/E-DiA by incubating the washed monolayer with 2 mL of
BacVector.RTM. Insect media containing 30 .mu.L of 20 mg/mL X-Gluc
Solution (EMD Biosciences-Novagen, Madison, Wis.) for approximately
2 hours in a 28.degree. C. incubator.
[0284] To prepare a seed stock of virus, elute a recombinant virus
by transferring a plug containing a recombinant virus plaque with a
sterile Pasteur pipet to 1 mL BacVector.RTM. Insect media (EMD
Biosciences-Novagen, Madison, Wis.) in a sterile screw-cap vial.
Incubate the vial for approximately 16 hours at 4.degree. C.
Approximately 5.times.10.sup.5 Sf9 cells are plated in T-25 flask
containing 5 mL of BacVector.RTM. Insect media (EMD
Biosciences-Novagen, Madison, Wis.) and are incubated for
approximately 20 minutes in a 28.degree. C. incubator. Remove the
media and add 300 .mu.L of eluted recombinant virus. After one
hour, add 5 mL BacVector.RTM. Insect media containing 5% bovine
serum albumin to the transfected cells and transfer the cells to a
28.degree. C. incubator for 3-5 days until the majority of cells
become unattached and unhealthy. The virus is harvested by
transferring the media to 15 mL snap-cap tubes and centrifuging
tubes at 1000.times. g for 5 minutes to remove debris. The
clarified supernatant is transferred to fresh 15 mL snap-cap tubes
and are stored at 4.degree. C.
[0285] To prepare a high titer stock of virus, approximately
2.times.10.sup.7 Sf9 cells are plated in T-75 flask containing 10
mL of BacVector.RTM. Insect media (EMD Biosciences-Novagen,
Madison, Wis.) and are incubated for approximately 20 minutes in a
28.degree. C. incubator. Remove the media and add 500 .mu.L of
virus seed stock. After one hour, add 10 mL BacVector.RTM. Insect
media containing 5% bovine serum albumin to the transfected cells
and transfer the cells to a 28.degree. C. incubator for 3-5 days
until the majority of cells become unattached and unhealthy. The
virus is harvested by transferring the media to 15 mL snap-cap
tubes and centrifuging tubes at 1000.times. g for 5 minutes to
remove debris. The clarified supernatant is transferred to fresh 15
mL snap-cap tubes and are stored at 4.degree. C. High titer virus
stocks should contain approximately 2.times.10.sup.8 to
3.times.10.sup.9 pfu of baculovirus.
[0286] To express gp64-BoNT/E-DiA using a baculoviral expression
system, about 1.25.times.10.sup.8 Sf9 cells are seeded in a 1 L
flask containing 250 mL of BacVector.RTM. Insect media and are
grown in an orbital shaker (150 rpm) to a cell density of
approximately 5.times.10.sup.8. The culture is inoculated with
approximately 2.5.times.10.sup.9 of high titer stock recombinant
baculovirus and incubated for approximately 48 hours in a
28.degree. C. orbital shaker (150 rpm). Media is harvested by
transferring the media to tubes and centrifuging tubes at
500.times. g for 5 minutes to remove debris. Media samples are
added to 2.times.LDS Sample Buffer (Invitrogen, Inc, Carlsbad,
Calif.) and expression is measured by Western blot analysis (as
described in Example 8) using either anti-BoNT/A or anti-His
antibodies in order to identify baculoviral stocks expressing
BoNT/E-DiA. A similar procedure can be used to express a pBACgus3
construct encoding BoNT/E-DiB, BoNT/E-DiF, BoNT/E-Ba, BoNT/E-DiT,
BoNT/B-DiA, BoNT/C1-DiA, BoNT/D-DiA, BoNT/F-DiA, BoNT/G-DiA,
TeNT-DiA, BaNT-DiA, and BuNT-DiA.
[0287] BoNT/E-DiA is purified using the IMAC procedure, as
described in Example 3. Expression from each culture is evaluated
by a Bradford dye assay, polyacrylamide gel electrophoresis and
Western blot analysis (as described in Example 3) in order to
determine the amounts of BoNT/E-DiA produced.
Example 6
Expression of Modified Clostridial Toxins in a Mammalian Cell
[0288] The following example illustrates a procedure useful for
expressing any of the modified Clostridial toxins disclosed in the
present specification in a mammalian cell.
[0289] To construct a suitable mammalian expression construct
encoding a modified Clostridial toxin, restriction endonuclease
sites suitable for cloning an operably linked polynucleotide
molecule into a pSecTag2 vector (Invitrogen, Inc, Carlsbad, Calif.)
are incorporated into the 5'- and 3' ends of the polynucleotide
molecule encoding BoNT/E-DiA of SEQ ID NO: 50. This polynucleotide
molecule is synthesized and a pUCBHB1/BoNT/E-DiA construct is
obtained as described in Example 1. This construct is digested with
restriction enzymes that 1) will excise the insert containing the
open reading frame encoding BoNT/E-DiA; and 2) enable this insert
to be operably-linked to a pSecTag2 vector. This insert is
subcloned using a T4 DNA ligase procedure into a pSecTag2 vector
that is digested with appropriate restriction endonucleases to
yield pSecTag2/BoNT/E-DiA. The ligation mixture is transformed into
chemically competent E. coli DH5.alpha. cells (Invitrogen, Inc,
Carlsbad, Calif.) using a heat shock method, plated on 1.5%
Luria-Bertani agar plates (pH 7.0) containing 100 .mu.g/mL of
Ampicillin, and placed in a 37.degree. C. incubator for overnight
growth. Bacteria containing expression constructs are identified as
Ampicillin resistant colonies. Candidate constructs are isolated
using an alkaline lysis plasmid mini-preparation procedure and
analyzed by restriction endonuclease digest mapping to determine
the presence and orientation of the insert. This cloning strategy
yielded a pSecTag2 expression construct comprising the
polynucleotide molecule encoding the BoNT/E-DiA of SEQ ID NO: 50
operably-linked to a carboxyl-terminal c-myc and polyhistidine
binding peptides.
[0290] A similar cloning strategy is used to make pSecTag2
expression constructs encoding BoNT/E-DiB, BoNT/E-DiF, BoNT/E-Ba,
BoNT/E-DiT, BoNT/B-DiA, BoNT/C1-DiA, BoNT/D-DiA, BoNT/F-DiA,
BoNT/G-DiA, TeNT-DiA, BaNT-DiA, and BuNT-DiA.
[0291] To transiently express modified Clostridial toxin in a cell
line, about 1.5.times.10.sup.5 SH-SY5Y cells are plated in a 35 mm
tissue culture dish containing 3 mL of complete Dulbecco's Modified
Eagle Media (DMEM), supplemented with 10% fetal bovine serum (FBS),
1.times. penicillin/streptomycin solution (Invitrogen, Inc,
Carlsbad, Calif.) and 1.times.MEM non-essential amino acids
solution (Invitrogen, Inc, Carlsbad, Calif.), and grown in a
37.degree. C. incubator under 5% carbon dioxide until cells reach a
density of about 5.times.10.sup.5 cells/ml (6-16 hours). A 500
.mu.L transfection solution is prepared by adding 250 .mu.L of
OPTI-MEM Reduced Serum Medium containing 15 .mu.L of LipofectAmine
2000 (Invitrogen, Carlsbad, Calif.) incubated at room temperature
for 5 minutes to 250 .mu.L of OPTI-MEM Reduced Serum Medium
containing 5 .mu.g of a pSecTag2 expression construct encoding a
modified Clostridial toxin, such as, e.g., pSecTag2/BoNT/E-DiA.
This transfection is incubated at room temperature for
approximately 20 minutes. The complete, supplemented DMEM media is
replaced with 2 mL of OPTI-MEM Reduced Serum Medium and the 500
.mu.L transfection solution is added to the SH-SY5Y cells and the
cells are incubated in a 37.degree. C. incubator under 5% carbon
dioxide for approximately 6 to 18 hours. Transfection media is
replaced with 3 mL of fresh complete, supplemented DMEM and the
cells are incubated in a 37.degree. C. incubator under 5% carbon
dioxide for 48 hours. Both media and cells are collected for
expression analysis of BoNT/E-DiA. Media is harvested by
transferring the media to 15 mL snap-cap tubes and centrifuging
tubes at 500.times. g for 5 minutes to remove debris. Cells are
harvested by rinsing cells once with 3.0 mL of 100 mM
phosphate-buffered saline, pH 7.4 and lysing cells with a buffer
containing 62.6 mM 2-amino-2-hydroxymethyl-1,3-propanediol
hydrochloric acid (Tris-HCl), pH 6.8 and 2% sodium lauryl sulfate
(SDS). Both media and cell samples are added to 2.times.LDS Sample
Buffer (Invitrogen, Inc, Carlsbad, Calif.) and expression is
measured by Western blot analysis (as described in Example 5) using
either anti-BoNT/E, anti-c-myc or anti-His antibodies in order to
identify pSecTag2 constructs expressing BoNT/E-DiA. A similar
procedure can be used to transiently express a pSecTag2 construct
encoding BoNT/E-DiB, BoNT/E-DiF, BoNT/E-Ba, BoNT/E-DiT, BoNT/B-DiA,
BoNT/C1-DiA, BoNT/D-DiA, BoNT/F-DiA, BoNT/G-DiA, TeNT-DiA,
BaNT-DiA, and BuNT-DiA.
[0292] To generate a stably-integrated cell line expressing a
modified Clostridial toxin, approximately 1.5.times.10.sup.5SH-SY5Y
cells are plated in a 35 mm tissue culture dish containing 3 mL of
complete DMEM, supplemented with 10% FBS, 1.times.
penicillin/streptomycin solution (Invitrogen, Inc, Carlsbad,
Calif.) and 1.times.MEM non-essential amino acids solution
(Invitrogen, Inc, Carlsbad, Calif.), and grown in a 37.degree. C.
incubator under 5% carbon dioxide until cells reach a density of
about 5.times.10.sup.5 cells/ml (6-16 hours). A 500 .mu.L
transfection solution is prepared by adding 250 .mu.L of OPTI-MEM
Reduced Serum Medium containing 15 .mu.L of LipofectAmine 2000
(Invitrogen, Carlsbad, Calif.) incubated at room temperature for 5
minutes to 250 .mu.L of OPTI-MEM Reduced Serum Medium containing 5
.mu.g of a pSecTag2 expression construct encoding a modified
Clostridial toxin, such as, e.g., pSecTag2/BoNT/E-DiA. This
transfection solution is incubated at room temperature for
approximately 20 minutes. The complete, supplemented DMEM media is
replaced with 2 mL of OPTI-MEM Reduced Serum Medium and the 500
.mu.L transfection solution is added to the SH-SY5Y cells and the
cells are incubated in a 37.degree. C. incubator under 5% carbon
dioxide for approximately 6 to 18 hours. Transfection media is
replaced with 3 mL of fresh complete, supplemented DMEM and cells
are incubated in a 37.degree. C. incubator under 5% carbon dioxide
for approximately 48 hours. Media is replaced with 3 mL of fresh
complete DMEM, containing approximately 5 .mu.g/mL of Zeocin.TM.,
10% FBS, 1.times. penicillin/streptomycin solution (Invitrogen,
Inc, Carlsbad, Calif.) and 1.times.MEM non-essential amino acids
solution (Invitrogen, Inc, Carlsbad, Calif.). Cells are incubated
in a 37.degree. C. incubator under 5% carbon dioxide for
approximately 3-4 weeks, with old media being replaced with fresh
Zeocin.TM.-selective, complete, supplemented DMEM every 4 to 5
days. Once Zeocin.TM.-resistant colonies are established, resistant
clones are replated to new 35 mm culture plates containing fresh
complete DMEM, supplemented with approximately 5 .mu.g/mL of
Zeocin.TM., 10% FBS, 1.times. penicillin/streptomycin solution
(Invitrogen, Inc, Carlsbad, Calif.) and 1.times.MEM non-essential
amino acids solution (Invitrogen, Inc, Carlsbad, Calif.), until
these cells reach a density of 6 to 20.times.10.sup.5 cells/mL. To
test for expression of BoNT/E-DiA from SH-SY5Y cell lines that have
stably-integrated a pSecTag2/BoNT/E-DiA, approximately
1.5.times.10.sup.5 SH-SY5Y cells from each cell line are plated in
a 35 mm tissue culture dish containing 3 mL of Zeocin.TM.
selective, complete, supplemented DMEM and grown in a 37.degree. C.
incubator under 5% carbon dioxide until cells reach a density of
about 5.times.10.sup.5 cells/ml (6-16 hours). Media is replaced
with 3 mL of fresh Zeocin.TM.-selective, complete, supplemented
DMEM and cells are incubated in a 37.degree. C. incubator under 5%
carbon dioxide for 48 hours. Both media and cells are collected for
expression analysis of BoNT/E-DiA-c-myc-His. Media is harvested by
transferring the media to 15 mL snap-cap tubes and centrifuging
tubes at 500.times. g for 5 minutes to remove debris. Cells are
harvest by rinsing cells once with 3.0 mL of 100 mM
phosphate-buffered saline, pH 7.4 and lysing cells with a buffer
containing 62.6 mM 2-amino-2-hydroxymethyl-1,3-propanediol
hydrochloric acid (Tris-HCl), pH 6.8 and 2% sodium lauryl sulfate
(SDS). Both media and cell samples are added to 2.times.LDS Sample
Buffer (Invitrogen, Inc, Carlsbad, Calif.) and expression is
measured by Western blot analysis (as described in Example 5) using
either anti-BoNT/A, anti-c-myc or anti-His antibodies in order to
identify SH-SY5Y cell lines expressing BoNT/E-DiA. The established
SH-SY5Y cell line showing the highest expression level of
BoNT/E-DiA is selected for large-scale expression using 3 L flasks.
Procedures for large-scale expression are as outlined above except
the starting volume is approximately 800-1000 mL of complete DMEM
and concentrations of all reagents are proportionally increased for
this volume. A similar procedure can be used to stably express a
pSecTag2 construct encoding BoNT/E-DiB, BoNT/E-DiF, BoNT/E-Ba,
BoNT/E-DiT, BoNT/B-DiA, BoNT/C1-DiA, BoNT/D-DiA, BoNT/F-DiA,
BoNT/G-DiA, TeNT-DiA, BaNT-DiA, and BuNT-DiA.
[0293] BoNT/E-DiA is purified using the IMAC procedure, as
described in Example 3. Expression from each culture is evaluated
by a Bradford dye assay, polyacrylamide gel electrophoresis and
Western blot analysis (as described in Example 3) in order to
determine whether the amounts of BoNT/E-DiA produced.
Example 7
Construction of Clostridial Toxins for Recombinant Expression
[0294] A polynucleotide molecule based on BoNT/A (SEQ ID NO: 1)
will be synthesized and cloned into a pUCBHB1 vector as described
in Example 1. If so desired, expression optimization to a different
organism, such as, e.g., a bacteria, a yeast strain, an insect
cell-line or a mammalian cell line, can be done as described above,
see, e.g., Steward, supra, (Feb. 2, 2006); and Steward, supra,
(Feb. 16, 2006). A similar cloning strategy will be used to make
pUCBHB1 cloning constructs BoNT/B (SEQ ID NO: 2), BoNT/C1 (SEQ ID
NO: 3), BoNT/D (SEQ ID NO: 4), BoNT/E (SEQ ID NO: 5), BoNT/F (SEQ
ID NO: 6), BoNT/G (SEQ ID NO: 7), TeNT (SEQ ID NO: 8), BaNT (SEQ ID
NO: 9), and BuNT (SEQ ID NO: 10).
[0295] To construct pET29/BoNT/A, a pUCBHB1/BoNT/A construct will
be digested with restriction endonucleases that 1) will excise the
polynucleotide molecule encoding the open reading frame of BoNT/A;
and 2) will enable this polynucleotide molecule to be
operably-linked to a pET29 vector (EMD Biosciences-Novagen,
Madison, Wis.). This insert will be subcloned using a T4 DNA ligase
procedure into a pET29 vector that is digested with appropriate
restriction endonucleases to yield pET29/BoNT/A. The ligation
mixture will be transformed into chemically competent E. coli DH5a
cells (Invitrogen, Inc, Carlsbad, Calif.) using a heat shock
method, will be plated on 1.5% Luria-Bertani agar plates (pH 7.0)
containing 50 .mu.g/mL of Kanamycin, and will be placed in a
37.degree. C. incubator for overnight growth. Bacteria containing
expression constructs will be identified as Kanamycin resistant
colonies. Candidate constructs will be isolated using an alkaline
lysis plasmid mini-preparation procedure and will be analyzed by
restriction endonuclease digest mapping to determine the presence
and orientation of the insert. This cloning strategy will yield a
pET29 expression construct comprising the polynucleotide molecule
encoding the BoNT/A operably-linked to a carboxyl terminal
polyhistidine affinity binding peptide. A similar cloning strategy
will be used to make pET29 expression constructs for other modified
BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G-TEV, TeNT-TEV,
BaNT, or BuNT.
Example 8
Expression and Purification of Recombinant Clostridial Toxins in a
Bacterial Cell
[0296] The following example illustrates a procedure useful for
recombinantly expressing any of the Clostridial toxins disclosed in
the present specification in a bacterial cell.
[0297] An expression construct, such as, e.g., pET29/BoNT/A, see,
e.g., Example 7 is introduced into chemically competent E. coli
BL21 (DE3) cells (Invitrogen, Inc, Carlsbad, Calif.) using a
heat-shock transformation protocol. The heat-shock reaction is
plated onto 1.5% Luria-Bertani agar plates (pH 7.0) containing 50
.mu.g/mL of Kanamycin and is placed in a 37.degree. C. incubator
for overnight growth. Kanamycin-resistant colonies of transformed
E. coli containing the expression construct, such as, e.g.,
pET29/BoNT/iA are used to inoculate a baffled flask containing 3.0
mL of PA-0.5G media containing 50 .mu.g/mL of Kanamycin which is
then placed in a 37.degree. C. incubator, shaking at 250 rpm, for
overnight growth. The resulting overnight starter culture is in
turn used to inoculate a 3 L baffled flask containing ZYP-5052
autoinducing media containing 50 .mu.g/mL of Kanamycin at a
dilution of 1:1000. Culture volumes ranged from about 600 mL (20%
flask volume) to about 750 mL (25% flask volume). These cultures
are grown in a 37.degree. C. incubator shaking at 250 rpm for
approximately 5.5 hours and are then transferred to a 16.degree. C.
incubator shaking at 250 rpm for overnight expression. Cells are
harvested by centrifugation (4,000 rpm at 4.degree. C. for 20-30
minutes) and are used immediately, or stored dry at -80.degree. C.
until needed.
[0298] Recombinantly-expressed BoNT/A is purified using the IMAC
procedure, as described in Example 3. Expression from each culture
is evaluated by a Bradford dye assay, polyacrylamide gel
electrophoresis and Western blot analysis (as described in Example
3) in order to determine the amounts of recombinantly-expressed
BoNT/A produced.
[0299] To activate purified, recombinantly-expressed BoNT/A,
approximately 30 .mu.g of purified, recombinantly-expressed BoNT/A
will be incubated with 3 .mu.g of di-chain loop protease of SEQ ID
NO: 33 in 20 mM Tris-HCl, pH 8.0 with 200 mM NaCl. Following
incubation at 23.degree. C. for 2 hours, the nicking reaction will
be quenched by addition of 1.times. Protease Inhibitor Cocktail Set
III (CalBiochem; inhibitor contains 1 mM AEBSF, 0.8 .mu.M
Aprotinin, 50 .mu.M Bestatin, 15 .mu.M E-64, 20 .mu.M Leupeptin,
and 10 .mu.M Pepstatin A). The samples may be flash frozen in
liquid nitrogen and immediately stored at -80.degree. C.
[0300] Although aspects of the present invention have been
described with reference to the disclosed embodiments, one skilled
in the art will readily appreciate that the specific examples
disclosed are only illustrative of these aspects and in no way
limit the present invention. Various modifications can be made
without departing from the spirit of the present invention.
Sequence CWU 1
1
5811296PRTClostridium botulinum serotype A 1Met Pro Phe Val Asn Lys
Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly1 5 10 15 Val Asp Ile Ala
Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30 Val Lys
Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45
Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50
55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser
Thr65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys
Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu
Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser
Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile
Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 Arg Ser Glu Glu Leu
Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile145 150 155 160 Ile Gln
Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175
Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180
185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu
Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu
Ala His Glu 210 215 220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile
Ala Ile Asn Pro Asn225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn
Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val Ser Phe Glu Glu
Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 Phe Ile Asp Ser
Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn 275 280 285 Lys Phe
Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290 295 300
Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys305
310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp
Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile
Tyr Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn
Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val Phe Lys Ile
Asn Ile Val Pro Lys Val Asn Tyr 370 375 380 Thr Ile Tyr Asp Gly Phe
Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn385 390 395 400 Phe Asn Gly
Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415 Lys
Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425
430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys
435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu
Phe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn
Lys Gly Glu Glu465 470 475 480 Ile Thr Ser Asp Thr Asn Ile Glu Ala
Ala Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile Gln Gln Tyr Tyr
Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 Glu Asn Ile Ser Ile
Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525 Glu Leu Met
Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540 Leu
Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu545 550
555 560 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala
Leu 565 570 575 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp
Tyr Val Lys 580 585 590 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe
Leu Gly Trp Val Glu 595 600 605 Gln Leu Val Tyr Asp Phe Thr Asp Glu
Thr Ser Glu Val Ser Thr Thr 610 615 620 Asp Lys Ile Ala Asp Ile Thr
Ile Ile Ile Pro Tyr Ile Gly Pro Ala625 630 635 640 Leu Asn Ile Gly
Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655 Ile Phe
Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670
Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675
680 685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn
Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp
Leu Ala Lys705 710 715 720 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys
Lys Met Lys Glu Ala Leu 725 730 735 Glu Asn Gln Ala Glu Ala Thr Lys
Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr Thr Glu Glu Glu
Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765 Leu Ser Ser Lys
Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780 Asn Lys
Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met785 790 795
800 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys
805 810 815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu
Ile Gly 820 825 830 Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr
Leu Ser Thr Asp 835 840 845 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp
Asn Gln Arg Leu Leu Ser 850 855 860 Thr Phe Thr Glu Tyr Ile Lys Asn
Ile Ile Asn Thr Ser Ile Leu Asn865 870 875 880 Leu Arg Tyr Glu Ser
Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895 Lys Ile Asn
Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910 Gln
Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920
925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser
930 935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu
Asn Asn945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn
Ser Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp
Thr Leu Gln Asp Thr Gln Glu 980 985 990 Ile Lys Gln Arg Val Val Phe
Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005 Asp Tyr Ile Asn
Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu 1010 1015 1020 Asn
Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln Lys Pro1025
1030 1035 1040Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile
Met Phe Lys 1045 1050 1055 Leu Asp Gly Cys Arg Asp Thr His Arg Tyr
Ile Trp Ile Lys Tyr Phe 1060 1065 1070 Asn Leu Phe Asp Lys Glu Leu
Asn Glu Lys Glu Ile Lys Asp Leu Tyr 1075 1080 1085 Asp Asn Gln Ser
Asn Ser Gly Ile Leu Lys Asp Phe Trp Gly Asp Tyr 1090 1095 1100 Leu
Gln Tyr Asp Lys Pro Tyr Tyr Met Leu Asn Leu Tyr Asp Pro Asn1105
1110 1115 1120Lys Tyr Val Asp Val Asn Asn Val Gly Ile Arg Gly Tyr
Met Tyr Leu 1125 1130 1135 Lys Gly Pro Arg Gly Ser Val Met Thr Thr
Asn Ile Tyr Leu Asn Ser 1140 1145 1150 Ser Leu Tyr Arg Gly Thr Lys
Phe Ile Ile Lys Lys Tyr Ala Ser Gly 1155 1160 1165 Asn Lys Asp Asn
Ile Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val 1170 1175 1180 Val
Val Lys Asn Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala1185
1190 1195 1200Gly Val Glu Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp
Val Gly Asn 1205 1210 1215 Leu Ser Gln Val Val Val Met Lys Ser Lys
Asn Asp Gln Gly Ile Thr 1220 1225 1230 Asn Lys Cys Lys Met Asn Leu
Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 Phe Ile Gly Phe
His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala Ser 1250 1255 1260 Asn
Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu Gly Cys1265
1270 1275 1280Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu
Arg Pro Leu 1285 1290 1295 21291PRTClostridium botulinum serotype B
2Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn1 5
10 15 Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly
Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile
Ile Pro Glu 35 40 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe
Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn Arg Asp Val Cys Glu Tyr
Tyr Asp Pro Asp Tyr Leu Asn65 70 75 80 Thr Asn Asp Lys Lys Asn Ile
Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95 Asn Arg Ile Lys Ser
Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 Ile Asn Gly
Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 Phe
Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135
140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile
Ile145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr
Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly
Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr Val
Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser Ile
Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile Leu
Met His Glu Leu Ile His Val Leu His Gly Leu Tyr225 230 235 240 Gly
Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250
255 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe
260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys
Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val
Asp Arg Leu Asn 290 295 300 Lys Val Leu Val Cys Ile Ser Asp Pro Asn
Ile Asn Ile Asn Ile Tyr305 310 315 320 Lys Asn Lys Phe Lys Asp Lys
Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr Ser Ile Asp
Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350 Met Phe Gly
Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 Thr
Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375
380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn
Ile385 390 395 400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln
Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys
Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val
Lys Ala Pro Gly Ile Cys Ile Asp 435 440 445 Val Asp Asn Glu Asp Leu
Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 Asp Asp Leu Ser
Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn465 470 475 480 Tyr
Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490
495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr
500 505 510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala
Ile Lys 515 520 525 Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr
Leu Tyr Ser Gln 530 535 540 Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser
Leu Thr Ser Ser Phe Asp545 550 555 560 Asp Ala Leu Leu Phe Ser Asn
Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr Ile Lys Thr Ala
Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 Trp Val Lys
Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 Asn
Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615
620 Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe
Glu625 630 635 640 Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu
Glu Phe Ile Pro 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Ala Phe
Leu Leu Glu Ser Tyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys
Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 Arg Asn Glu Lys Trp Ser
Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 Leu Ser Thr Val
Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr705 710 715 720 Lys
Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730
735 Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp
740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln
Ala Ile 755 760 765 Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val
Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile Pro Leu Ala Val Glu Lys
Leu Leu Asp Phe Asp Asn785 790 795 800 Thr Leu Lys Lys Asn Leu Leu
Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu Ile Gly Ser Ala
Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825 830 Lys Thr Ile
Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835 840 845 Leu
Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855
860 Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser
Gly865 870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu
Leu Asn Asp Lys 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn
Ser Lys Ile Arg Val Thr 900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn
Ser Val Phe Leu Asp Phe Ser Val 915 920 925 Ser Phe Trp Ile Arg Ile
Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn 930 935 940 Tyr Ile His Asn
Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser945 950 955 960 Gly
Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970
975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg
980 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr
Ile Thr 995 1000 1005 Asn
Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser 1010
1015 1020 Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly
Glu Ile1025 1030 1035 1040Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg
Thr Gln Phe Ile Trp Met 1045 1050 1055 Lys Tyr Phe Ser Ile Phe Asn
Thr Glu Leu Ser Gln Ser Asn Ile Glu 1060 1065 1070 Glu Arg Tyr Lys
Ile Gln Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp 1075 1080 1085 Gly
Asn Pro Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly 1090
1095 1100 Asn Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val
Gly Glu1105 1110 1115 1120Ile Leu Thr Arg Ser Lys Tyr Asn Gln Asn
Ser Lys Tyr Ile Asn Tyr 1125 1130 1135 Arg Asp Leu Tyr Ile Gly Glu
Lys Phe Ile Ile Arg Arg Lys Ser Asn 1140 1145 1150 Ser Gln Ser Ile
Asn Asp Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr 1155 1160 1165 Leu
Asp Phe Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys 1170
1175 1180 Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile
Ser Asp1185 1190 1195 1200Ser Asp Glu Phe Tyr Asn Thr Ile Gln Ile
Lys Glu Tyr Asp Glu Gln 1205 1210 1215 Pro Thr Tyr Ser Cys Gln Leu
Leu Phe Lys Lys Asp Glu Glu Ser Thr 1220 1225 1230 Asp Glu Ile Gly
Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile 1235 1240 1245 Val
Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu 1250
1255 1260 Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys
Asn Trp1265 1270 1275 1280Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr
Glu 1285 1290 31291PRTClostridium botulinum serotype C1 3Met Pro
Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15
Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20
25 30 Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro
Asp 35 40 45 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro
Pro Arg Val 50 55 60 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn
Tyr Leu Ser Thr Asp65 70 75 80 Ser Asp Lys Asp Pro Phe Leu Lys Glu
Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Ser Arg Glu Ile Gly
Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110 Asp Ile Pro Phe Pro
Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125 Phe Asp Val
Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 Asn
Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150
155 160 Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu
Thr 165 170 175 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu
Ser Ile Ile 180 185 190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser
Asn Ala Thr Asn Asp 195 200 205 Val Gly Glu Gly Arg Phe Ser Lys Ser
Glu Phe Cys Met Asp Pro Ile 210 215 220 Leu Ile Leu Met His Glu Leu
Asn His Ala Met His Asn Leu Tyr Gly225 230 235 240 Ile Ala Ile Pro
Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255 Phe Tyr
Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270
Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275
280 285 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg
Leu 290 295 300 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys
Tyr Ile Gly305 310 315 320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr
Arg Phe Val Val Glu Ser 325 330 335 Ser Gly Glu Val Thr Val Asn Arg
Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350 Glu Leu Thr Gln Ile Phe
Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 Val Gln Asn Arg
Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 Ala Asn
Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395
400 Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser
405 410 415 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu
Tyr Leu 420 425 430 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg
Ser Leu Tyr Asn 435 440 445 Lys Thr Leu Asp Cys Arg Glu Leu Leu Val
Lys Asn Thr Asp Leu Pro 450 455 460 Phe Ile Gly Asp Ile Ser Asp Val
Lys Thr Asp Ile Phe Leu Arg Lys465 470 475 480 Asp Ile Asn Glu Glu
Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495 Val Asp Gln
Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510 Asp
Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520
525 Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu
530 535 540 Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn
Val Glu545 550 555 560 Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala
Leu Asp Asn Ser Ala 565 570 575 Lys Val Tyr Thr Tyr Phe Pro Thr Leu
Ala Asn Lys Val Asn Ala Gly 580 585 590 Val Gln Gly Gly Leu Phe Leu
Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605 Phe Thr Thr Asn Ile
Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620 Val Ser Ala
Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn625 630 635 640
Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645
650 655 Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu
Gly 660 665 670 Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu
Ile Ile Lys 675 680 685 Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys
Arg Trp Lys Asp Ser 690 695 700 Tyr Glu Trp Met Met Gly Thr Trp Leu
Ser Arg Ile Ile Thr Gln Phe705 710 715 720 Asn Asn Ile Ser Tyr Gln
Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735 Ala Ile Lys Ala
Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750 Asp Lys
Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765
Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770
775 780 Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val
Ile785 790 795 800 Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala
Lys Leu Ile Asn 805 810 815 Leu Ile Asp Ser His Asn Ile Ile Leu Val
Gly Glu Val Asp Lys Leu 820 825 830 Lys Ala Lys Val Asn Asn Ser Phe
Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845 Phe Ser Tyr Thr Asn Asn
Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860 Phe Asn Asn Ile
Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys865 870 875 880 Asn
Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890
895 Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly
900 905 910 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn
Glu Asn 915 920 925 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile
Ser Phe Trp Ile 930 935 940 Arg Ile Asn Lys Trp Val Ser Asn Leu Pro
Gly Tyr Thr Ile Ile Asp945 950 955 960 Ser Val Lys Asn Asn Ser Gly
Trp Ser Ile Gly Ile Ile Ser Asn Phe 965 970 975 Leu Val Phe Thr Leu
Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn 980 985 990 Phe Ser Tyr
Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005
Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr Ile
1010 1015 1020 Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu
Thr Gly Ile1025 1030 1035 1040Asn Phe Ser Lys Thr Ile Thr Phe Glu
Ile Asn Lys Ile Pro Asp Thr 1045 1050 1055 Gly Leu Ile Thr Ser Asp
Ser Asp Asn Ile Asn Met Trp Ile Arg Asp 1060 1065 1070 Phe Tyr Ile
Phe Ala Lys Glu Leu Asp Gly Lys Asp Ile Asn Ile Leu 1075 1080 1085
Phe Asn Ser Leu Gln Tyr Thr Asn Val Val Lys Asp Tyr Trp Gly Asn
1090 1095 1100 Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr Met Val Asn Ile
Asp Tyr Leu1105 1110 1115 1120Asn Arg Tyr Met Tyr Ala Asn Ser Arg
Gln Ile Val Phe Asn Thr Arg 1125 1130 1135 Arg Asn Asn Asn Asp Phe
Asn Glu Gly Tyr Lys Ile Ile Ile Lys Arg 1140 1145 1150 Ile Arg Gly
Asn Thr Asn Asp Thr Arg Val Arg Gly Gly Asp Ile Leu 1155 1160 1165
Tyr Phe Asp Met Thr Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys
1170 1175 1180 Asn Glu Thr Met Tyr Ala Asp Asn His Ser Thr Glu Asp
Ile Tyr Ala1185 1190 1195 1200Ile Gly Leu Arg Glu Gln Thr Lys Asp
Ile Asn Asp Asn Ile Ile Phe 1205 1210 1215 Gln Ile Gln Pro Met Asn
Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe 1220 1225 1230 Lys Ser Asn
Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235 1240 1245
Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr Leu
1250 1255 1260 Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu
Glu Ser Thr1265 1270 1275 1280Ser Thr His Trp Gly Phe Val Pro Val
Ser Glu 1285 1290 41276PRTClostridium botulinum serotype D 4Met Thr
Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp1 5 10 15
Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20
25 30 Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro
Glu 35 40 45 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro
Pro Arg Pro 50 55 60 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser
Tyr Leu Ser Thr Asp65 70 75 80 Glu Gln Lys Asp Thr Phe Leu Lys Gly
Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Glu Arg Asp Ile Gly
Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110 Gly Ser Pro Phe Met
Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125 Phe Thr Arg
His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140 Ser
Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly145 150
155 160 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln
Gly 165 170 175 Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu
Ser Ile Leu 180 185 190 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser
Asp Val Thr Ser Asn 195 200 205 Gln Ser Ser Ala Val Leu Gly Lys Ser
Ile Phe Cys Met Asp Pro Val 210 215 220 Ile Ala Leu Met His Glu Leu
Thr His Ser Leu His Gln Leu Tyr Gly225 230 235 240 Ile Asn Ile Pro
Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255 Phe Phe
Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270
Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275
280 285 Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg
Leu 290 295 300 Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser
Asn Ile Asp305 310 315 320 Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr
Asn Phe Asp Lys Asp Asn 325 330 335 Thr Gly Asn Phe Val Val Asn Ile
Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350 Asp Leu Thr Asn Val Met
Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365 Val Lys Asn Arg
Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380 Ala Asn
Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn385 390 395
400 Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu
405 410 415 Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val
Asp Leu 420 425 430 Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser
Arg Asp Asp Ser 435 440 445 Thr Cys Ile Lys Val Lys Asn Asn Arg Leu
Pro Tyr Val Ala Asp Lys 450 455 460 Asp Ser Ile Ser Gln Glu Ile Phe
Glu Asn Lys Ile Ile Thr Asp Glu465 470 475 480 Thr Asn Val Gln Asn
Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile 485 490 495 Leu Asp Gly
Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu 500 505 510 Pro
Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val 515 520
525 Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr
530 535 540 Tyr Leu Glu Ser Gln Lys Leu Ser Asn Asn Val Glu Asn Ile
Thr Leu545 550 555 560 Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser
Asn Lys Ile Tyr Thr 565 570 575 Phe Leu Pro Ser Leu Ala Glu Lys Val
Asn Lys Gly Val Gln Ala Gly 580 585 590 Leu Phe Leu Asn Trp Ala Asn
Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605 Ile Met Lys Lys Asp
Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615 620 Ile Pro Tyr
Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg625 630 635 640
Gly Asn Phe Asn Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645
650 655 Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr
Phe 660 665 670 Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr
Ile Glu Asn 675 680 685 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp
Ser Tyr Gln Trp Met 690 695 700 Val Ser Asn Trp Leu Ser Arg Ile Thr
Thr Gln Phe Asn His Ile Asn705 710 715 720 Tyr Gln Met Tyr Asp Ser
Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala 725
730 735 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu
Asn 740 745 750 Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp
Val Lys Ile 755 760 765 Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile
Arg Glu Cys Ser Val 770 775 780 Thr Tyr Leu Phe Lys Asn Met Leu Pro
Lys Val Ile Asp Glu Leu Asn785 790 795 800 Lys Phe Asp Leu Arg Thr
Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805 810 815 His Asn Ile Ile
Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val 820 825 830 Asn Glu
Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 840 845
Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile 850
855 860 Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn Ala Leu
Val865 870 875 880 Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly
Asp Asn Val Gln 885 890 895 Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys
Leu Ser Ser Ser Gly Asp 900 905 910 Lys Ile Ile Val Asn Leu Asn Asn
Asn Ile Leu Tyr Ser Ala Ile Tyr 915 920 925 Glu Asn Ser Ser Val Ser
Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr 930 935 940 Asn Ser His Asn
Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser945 950 955 960 Gly
Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln 965 970
975 Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser
980 985 990 Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr
Ile Thr 995 1000 1005 Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile
Asn Gly Glu Leu Lys 1010 1015 1020 Gln Ser Gln Lys Ile Glu Asp Leu
Asp Glu Val Lys Leu Asp Lys Thr1025 1030 1035 1040Ile Val Phe Gly
Ile Asp Glu Asn Ile Asp Glu Asn Gln Met Leu Trp 1045 1050 1055 Ile
Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser Asn Glu Asp Ile 1060
1065 1070 Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn Val Ile Lys
Asp Tyr 1075 1080 1085 Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu Tyr
Tyr Ile Ile Asn Asp 1090 1095 1100 Asn Tyr Ile Asp Arg Tyr Ile Ala
Pro Glu Ser Asn Val Leu Val Leu1105 1110 1115 1120Val Gln Tyr Pro
Asp Arg Ser Lys Leu Tyr Thr Gly Asn Pro Ile Thr 1125 1130 1135 Ile
Lys Ser Val Ser Asp Lys Asn Pro Tyr Ser Arg Ile Leu Asn Gly 1140
1145 1150 Asp Asn Ile Ile Leu His Met Leu Tyr Asn Ser Arg Lys Tyr
Met Ile 1155 1160 1165 Ile Arg Asp Thr Asp Thr Ile Tyr Ala Thr Gln
Gly Gly Glu Cys Ser 1170 1175 1180 Gln Asn Cys Val Tyr Ala Leu Lys
Leu Gln Ser Asn Leu Gly Asn Tyr1185 1190 1195 1200Gly Ile Gly Ile
Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr 1205 1210 1215 Cys
Ser Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala 1220
1225 1230 Asp Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr
Thr Pro 1235 1240 1245 Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu
Ser Thr Ser Ser Phe 1250 1255 1260 Trp Lys Phe Ile Ser Arg Asp Pro
Gly Trp Val Glu1265 1270 1275 51252PRTClostridium botulinum
serotype E 5Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn
Asp Arg1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu
Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile
Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Thr Pro Gln Asp Phe His
Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr Asp
Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys65 70 75 80 Asp Arg Phe Leu
Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn Leu
Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110
Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115
120 125 Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile
Leu 130 135 140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu
Phe Glu Thr145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn
Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly Ser Ile Ala Ile Val
Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe Asn Asp Asn Ser
Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr Leu Met His
Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 Lys Gly
Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu225 230 235
240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly
245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp
Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser
Lys Leu Ser Lys 275 280 285 Val Gln Val Ser Asn Pro Leu Leu Asn Pro
Tyr Lys Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp Lys Asp
Ala Ser Gly Ile Tyr Ser Val Asn305 310 315 320 Ile Asn Lys Phe Asn
Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe Asp Leu
Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 Gly
Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360
365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe
370 375 380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro
Ile Thr385 390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe
Cys Lys Asn Ile Val 405 410 415 Ser Val Lys Gly Ile Arg Lys Ser Ile
Cys Ile Glu Ile Asn Asn Gly 420 425 430 Glu Leu Phe Phe Val Ala Ser
Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 Asn Thr Pro Lys Glu
Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 Glu Asn Asp
Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala465 470 475 480
Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485
490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln
His 500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala
Gln Lys Val 515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser
Ser Ile Asp Thr Ala 530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr Thr
Phe Phe Ser Ser Glu Phe Ile545 550 555 560 Asn Asn Val Asn Lys Pro
Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 Gln Gln Val Leu
Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 Val Asp
Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605
Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610
615 620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu
Leu625 630 635 640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser
Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys Asn Lys Val Ile Lys Ala
Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Lys Glu
Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 Trp Met Thr Lys Ile Asn
Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 Tyr Gln Ala Leu
Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu705 710 715 720 Ser
Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730
735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser
740 745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser
Ile Ser 755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn
Lys Leu Arg Glu 770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu
Asn Tyr Ile Ile Gln His785 790 795 800 Gly Ser Ile Leu Gly Glu Ser
Gln Gln Glu Leu Asn Ser Met Val Thr 805 810 815 Asp Thr Leu Asn Asn
Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 Asp Lys Ile
Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845 Ser
Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855
860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr
Lys865 870 875 880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn
Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile
Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn Phe Ser Ile Ser Phe Trp
Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys Ile Val Asn Val Asn
Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 Asp Asn Asn Ser
Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile945 950 955 960 Trp
Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970
975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe
980 985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr
Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn
Leu Gly Asn Ile His 1010 1015 1020 Val Ser Asp Asn Ile Leu Phe Lys
Ile Val Asn Cys Ser Tyr Thr Arg1025 1030 1035 1040Tyr Ile Gly Ile
Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu 1045 1050 1055 Thr
Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu 1060
1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp Lys Glu Tyr
Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys Pro Asn Asn Phe Ile Asp
Arg Arg Lys Asp Ser 1090 1095 1100 Thr Leu Ser Ile Asn Asn Ile Arg
Ser Thr Ile Leu Leu Ala Asn Arg1105 1110 1115 1120Leu Tyr Ser Gly
Ile Lys Val Lys Ile Gln Arg Val Asn Asn Ser Ser 1125 1130 1135 Thr
Asn Asp Asn Leu Val Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe 1140
1145 1150 Val Ala Ser Lys Thr His Leu Phe Pro Leu Tyr Ala Asp Thr
Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys Thr Ile Lys Ile Ser Ser
Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln Val Val Val Met Asn Ser
Val Gly Asn Asn Cys Thr Met Asn1185 1190 1195 1200Phe Lys Asn Asn
Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala 1205 1210 1215 Asp
Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His 1220
1225 1230 Thr Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu
His Gly 1235 1240 1245 Trp Gln Glu Lys 1250 61274PRTClostridium
botulinum serotype F 6Met Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn
Asp Pro Val Asn Asp1 5 10 15 Asp Thr Ile Leu Tyr Met Gln Ile Pro
Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr Tyr Lys Ala Phe Glu Ile
Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45 Arg Asn Thr Ile Gly
Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60 Leu Lys Asn
Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr65 70 75 80 Asp
Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys 85 90
95 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gln Glu Ile Ser
100 105 110 Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp
Glu Phe 115 120 125 Ser Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys
Leu Ser Thr Asn 130 135 140 Val Glu Ser Ser Met Leu Leu Asn Leu Leu
Val Leu Gly Ala Gly Pro145 150 155 160 Asp Ile Phe Glu Ser Cys Cys
Tyr Pro Val Arg Lys Leu Ile Asp Pro 165 170 175 Asp Val Val Tyr Asp
Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile 180 185 190 Val Thr Phe
Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 195 200 205 Gly
His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser 210 215
220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala
Arg225 230 235 240 Gly Val Thr Tyr Glu Glu Thr Ile Glu Val Lys Gln
Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu
Phe Leu Thr Phe Gly Gly 260 265 270 Gln Asp Leu Asn Ile Ile Thr Ser
Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285 Asn Leu Leu Ala Asn Tyr
Glu Lys Ile Ala Thr Arg Leu Ser Glu Val 290 295 300 Asn Ser Ala Pro
Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr Phe305 310 315 320 Gln
Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330
335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr
340 345 350 Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn
Thr Tyr 355 360 365 Phe Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu
Leu Asp Asp Asp 370 375 380 Ile Tyr Thr Val Ser Glu Gly Phe Asn Ile
Gly Asn Leu Ala Val Asn385 390 395 400 Asn Arg Gly Gln Ser Ile Lys
Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415 Pro Asp Lys Gly Leu
Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val 420 425 430 Ile Pro Arg
Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val 435 440 445 Asn
Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu 450 455
460 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu
Asn465 470 475 480 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu
Asp Tyr Asn Ser 485 490 495 Gln Thr Ile Pro Gln Ile Ser Asn Arg Thr
Leu Asn Thr Leu Val Gln 500
505 510 Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu
Ile 515 520 525 Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr
Leu His Ala 530 535 540 Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser
Leu Thr Ser Ser Ile545 550 555 560 Asp Thr Ala Leu Leu Glu Glu Ser
Lys Asp Ile Phe Phe Ser Ser Glu 565 570 575 Phe Ile Asp Thr Ile Asn
Lys Pro Val Asn Ala Ala Leu Phe Ile Asp 580 585 590 Trp Ile Ser Lys
Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln Lys 595 600 605 Ser Thr
Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Val 610 615 620
Gly Leu Ala Leu Asn Ile Ile Ile Glu Ala Glu Lys Gly Asn Phe Glu625
630 635 640 Glu Ala Phe Glu Leu Leu Gly Val Gly Ile Leu Leu Glu Phe
Val Pro 645 650 655 Glu Leu Thr Ile Pro Val Ile Leu Val Phe Thr Ile
Lys Ser Tyr Ile 660 665 670 Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile
Lys Ala Ile Asn Asn Ser 675 680 685 Leu Ile Glu Arg Glu Ala Lys Trp
Lys Glu Ile Tyr Ser Trp Ile Val 690 695 700 Ser Asn Trp Leu Thr Arg
Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu705 710 715 720 Gln Met Tyr
Gln Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr Ala 725 730 735 Ile
Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740 745
750 Glu Ser Glu Tyr Asn Ile Asn Asn Ile Glu Glu Glu Leu Asn Lys Lys
755 760 765 Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu
Ser Ser 770 775 780 Ile Ser Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys
Val Gly Lys Leu785 790 795 800 Lys Lys Tyr Asp Asn His Val Lys Ser
Asp Leu Leu Asn Tyr Ile Leu 805 810 815 Asp His Arg Ser Ile Leu Gly
Glu Gln Thr Asn Glu Leu Ser Asp Leu 820 825 830 Val Thr Ser Thr Leu
Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser Tyr 835 840 845 Thr Asn Asp
Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr Lys Lys 850 855 860 Ile
Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe865 870
875 880 Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asn
Val 885 890 895 Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr
Asn Ser Arg 900 905 910 Leu Ser Glu Val Asn Ile Ala Gln Asn Asn Asp
Ile Ile Tyr Asn Ser 915 920 925 Arg Tyr Gln Asn Phe Ser Ile Ser Phe
Trp Val Arg Ile Pro Lys His 930 935 940 Tyr Lys Pro Met Asn His Asn
Arg Glu Tyr Thr Ile Ile Asn Cys Met945 950 955 960 Gly Asn Asn Asn
Ser Gly Trp Lys Ile Ser Leu Arg Thr Val Arg Asp 965 970 975 Cys Glu
Ile Ile Trp Thr Leu Gln Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990
Leu Ile Phe Arg Tyr Glu Glu Leu Asn Arg Ile Ser Asn Tyr Ile Asn 995
1000 1005 Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn
Ser Arg 1010 1015 1020 Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys
Ser Ile Ser Asn Leu1025 1030 1035 1040Gly Asp Ile His Val Ser Asp
Asn Ile Leu Phe Lys Ile Val Gly Cys 1045 1050 1055 Asp Asp Glu Thr
Tyr Val Gly Ile Arg Tyr Phe Lys Val Phe Asn Thr 1060 1065 1070 Glu
Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr Ser Asn Glu Pro Asp 1075
1080 1085 Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn Tyr Leu Leu Tyr
Asn Lys 1090 1095 1100 Lys Tyr Tyr Leu Phe Asn Leu Leu Arg Lys Asp
Lys Tyr Ile Thr Leu1105 1110 1115 1120Asn Ser Gly Ile Leu Asn Ile
Asn Gln Gln Arg Gly Val Thr Glu Gly 1125 1130 1135 Ser Val Phe Leu
Asn Tyr Lys Leu Tyr Glu Gly Val Glu Val Ile Ile 1140 1145 1150 Arg
Lys Asn Gly Pro Ile Asp Ile Ser Asn Thr Asp Asn Phe Val Arg 1155
1160 1165 Lys Asn Asp Leu Ala Tyr Ile Asn Val Val Asp Arg Gly Val
Glu Tyr 1170 1175 1180 Arg Leu Tyr Ala Asp Thr Lys Ser Glu Lys Glu
Lys Ile Ile Arg Thr1185 1190 1195 1200Ser Asn Leu Asn Asp Ser Leu
Gly Gln Ile Ile Val Met Asp Ser Ile 1205 1210 1215 Gly Asn Asn Cys
Thr Met Asn Phe Gln Asn Asn Asn Gly Ser Asn Ile 1220 1225 1230 Gly
Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235
1240 1245 Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe
Trp Ser 1250 1255 1260 Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu1265
1270 71297PRTClostridium botulinum serotype G 7Met Pro Val Asn Ile
Lys Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn1 5 10 15 Asp Asp Ile
Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30 Tyr
Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu 35 40
45 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly
50 55 60 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr
Leu Lys65 70 75 80 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met
Ile Lys Leu Phe 85 90 95 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln
Arg Leu Leu Asp Met Ile 100 105 110 Val Asp Ala Ile Pro Tyr Leu Gly
Asn Ala Ser Thr Pro Pro Asp Lys 115 120 125 Phe Ala Ala Asn Val Ala
Asn Val Ser Ile Asn Lys Lys Ile Ile Gln 130 135 140 Pro Gly Ala Glu
Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile145 150 155 160 Phe
Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile 165 170
175 Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met
180 185 190 Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val
Gln Glu 195 200 205 Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr
Phe Ala Asp Pro 210 215 220 Ala Leu Thr Leu Met His Glu Leu Ile His
Val Leu His Gly Leu Tyr225 230 235 240 Gly Ile Lys Ile Ser Asn Leu
Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255 Phe Met Gln His Ser
Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly His
Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile 275 280 285 Tyr
Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290 295
300 Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr
Lys305 310 315 320 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp
Pro Asn Gly Lys 325 330 335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys
Leu Tyr Lys Ala Leu Met 340 345 350 Phe Gly Phe Thr Glu Thr Asn Leu
Ala Gly Glu Tyr Gly Ile Lys Thr 355 360 365 Arg Tyr Ser Tyr Phe Ser
Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys 370 375 380 Leu Leu Asp Asn
Thr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala385 390 395 400 Ser
Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn Lys Ala Val Asn 405 410
415 Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg
420 425 430 Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys
Ser Glu 435 440 445 Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe
Ile Ala Asn Lys 450 455 460 Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala
Glu Thr Ile Ala Tyr Asn465 470 475 480 Thr Gln Asn Asn Thr Ile Glu
Asn Asn Phe Ser Ile Asp Gln Leu Ile 485 490 495 Leu Asp Asn Asp Leu
Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr 500 505 510 Glu Pro Phe
Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys 515 520 525 Gln
Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535
540 Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln
Leu545 550 555 560 Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn
Lys Val Tyr Thr 565 570 575 Phe Phe Ser Thr Asn Leu Val Glu Lys Ala
Asn Thr Val Val Gly Ala 580 585 590 Ser Leu Phe Val Asn Trp Val Lys
Gly Val Ile Asp Asp Phe Thr Ser 595 600 605 Glu Ser Thr Gln Lys Ser
Thr Ile Asp Lys Val Ser Asp Val Ser Ile 610 615 620 Ile Ile Pro Tyr
Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala625 630 635 640 Lys
Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu 645 650
655 Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr
660 665 670 Leu Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr
Ile Ser 675 680 685 Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp
Met Tyr Gly Leu 690 695 700 Ile Val Ser Gln Trp Leu Ser Thr Val Asn
Thr Gln Phe Tyr Thr Ile705 710 715 720 Lys Glu Arg Met Tyr Asn Ala
Leu Asn Asn Gln Ser Gln Ala Ile Glu 725 730 735 Lys Ile Ile Glu Asp
Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met 740 745 750 Asn Ile Asn
Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser 755 760 765 Ile
Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser 770 775
780 Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys
Leu785 790 795 800 Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu
Glu Tyr Ile Asp 805 810 815 Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val
Asn Ile Leu Lys Ser Lys 820 825 830 Val Asn Arg His Leu Lys Asp Ser
Ile Pro Phe Asp Leu Ser Leu Tyr 835 840 845 Thr Lys Asp Thr Ile Leu
Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn 850 855 860 Ile Ser Ser Asn
Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu865 870 875 880 Ile
Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 885 890
895 Ile Phe Asn Asp Ile Gly Asn Gly Gln Phe Lys Leu Asn Asn Ser Glu
900 905 910 Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe Val Val Tyr
Asp Ser 915 920 925 Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg
Thr Pro Lys Tyr 930 935 940 Asn Asn Asn Asp Ile Gln Thr Tyr Leu Gln
Asn Glu Tyr Thr Ile Ile945 950 955 960 Ser Cys Ile Lys Asn Asp Ser
Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970 975 Arg Ile Ile Trp Thr
Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile 980 985 990 Phe Phe Glu
Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys 995 1000 1005
Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn Ile
1010 1015 1020 Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu
Asn Leu Asp1025 1030 1035 1040Arg Ile Asn Ser Ser Asn Asp Ile Asp
Phe Lys Leu Ile Asn Cys Thr 1045 1050 1055 Asp Thr Thr Lys Phe Val
Trp Ile Lys Asp Phe Asn Ile Phe Gly Arg 1060 1065 1070 Glu Leu Asn
Ala Thr Glu Val Ser Ser Leu Tyr Trp Ile Gln Ser Ser 1075 1080 1085
Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr
1090 1095 1100 Gln Tyr Tyr Leu Phe Asn Gln Gly Met Gln Asn Ile Tyr
Ile Lys Tyr1105 1110 1115 1120Phe Ser Lys Ala Ser Met Gly Glu Thr
Ala Pro Arg Thr Asn Phe Asn 1125 1130 1135 Asn Ala Ala Ile Asn Tyr
Gln Asn Leu Tyr Leu Gly Leu Arg Phe Ile 1140 1145 1150 Ile Lys Lys
Ala Ser Asn Ser Arg Asn Ile Asn Asn Asp Asn Ile Val 1155 1160 1165
Arg Glu Gly Asp Tyr Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu
1170 1175 1180 Ser Tyr Arg Val Tyr Val Leu Val Asn Ser Lys Glu Ile
Gln Thr Gln1185 1190 1195 1200Leu Phe Leu Ala Pro Ile Asn Asp Asp
Pro Thr Phe Tyr Asp Val Leu 1205 1210 1215 Gln Ile Lys Lys Tyr Tyr
Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu 1220 1225 1230 Cys Glu Lys
Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe 1235 1240 1245
Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys
1250 1255 1260 Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile
Asn Lys Leu1265 1270 1275 1280Arg Leu Gly Cys Asn Trp Gln Phe Ile
Pro Val Asp Glu Gly Trp Thr 1285 1290 1295 Glu81315PRTClostridium
teteni 8Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn
Asn1 5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly
Leu Asp Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile
Trp Ile Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu
Asp Phe Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu
Tyr Tyr Asp Pro Asn Tyr Leu Arg Thr65 70 75 80 Asp Ser Asp Lys Asp
Arg Phe Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys
Asn Asn Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn
Ala Ile Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120
125 Asp Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro
130 135 140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile
Ile Phe145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val
Arg Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro
Cys Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys
Pro Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile
Thr Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro
Ala Leu Leu Leu Met His Glu Leu Ile His Val Leu His225 230 235 240
Gly Leu Tyr Gly Met
Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250 255 Gln Glu Ile
Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu 260 265 270 Phe
Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp Ile Lys 275 280
285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys Ala Ile Ala Asn
290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp Pro Asn Ile Asp
Ile Asp305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln Gln Lys Tyr Gln
Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile Val Asn Glu Asp
Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met Tyr Gly Phe Thr
Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile Lys Thr Arg Leu
Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys 370 375 380 Ile Pro Asn
Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly Phe385 390 395 400
Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys Gly Gln Asn Met 405
410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val Asp Gly Ser Gly Leu
Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Lys Lys Ile Ile Pro Pro
Thr Asn Ile 435 440 445 Arg Glu Asn Leu Tyr Asn Arg Thr Ala Ser Leu
Thr Asp Leu Gly Gly 450 455 460 Glu Leu Cys Ile Lys Ile Lys Asn Glu
Asp Leu Thr Phe Ile Ala Glu465 470 475 480 Lys Asn Ser Phe Ser Glu
Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr 485 490 495 Asn Thr Lys Asn
Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile 500 505 510 Ile Val
Asp Tyr Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg 515 520 525
Thr Thr Pro Val Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser 530
535 540 Asn Ala Ala Ser Thr Ile Glu Ile His Asn Ile Asp Asp Asn Thr
Ile545 550 555 560 Tyr Gln Tyr Leu Tyr Ala Gln Lys Ser Pro Thr Thr
Leu Gln Arg Ile 565 570 575 Thr Met Thr Asn Ser Val Asp Asp Ala Leu
Ile Asn Ser Thr Lys Ile 580 585 590 Tyr Ser Tyr Phe Pro Ser Val Ile
Ser Lys Val Asn Gln Gly Ala Gln 595 600 605 Gly Ile Leu Phe Leu Gln
Trp Val Arg Asp Ile Ile Asp Asp Phe Thr 610 615 620 Asn Glu Ser Ser
Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser625 630 635 640 Thr
Ile Val Pro Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly 645 650
655 Tyr Glu Gly Asn Phe Ile Gly Ala Leu Glu Thr Thr Gly Val Val Leu
660 665 670 Leu Leu Glu Tyr Ile Pro Glu Ile Thr Leu Pro Val Ile Ala
Ala Leu 675 680 685 Ser Ile Ala Glu Ser Ser Thr Gln Lys Glu Lys Ile
Ile Lys Thr Ile 690 695 700 Asp Asn Phe Leu Glu Lys Arg Tyr Glu Lys
Trp Ile Glu Val Tyr Lys705 710 715 720 Leu Val Lys Ala Lys Trp Leu
Gly Thr Val Asn Thr Gln Phe Gln Lys 725 730 735 Arg Ser Tyr Gln Met
Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile 740 745 750 Lys Lys Ile
Ile Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys 755 760 765 Glu
Gln Ile Ala Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu 770 775
780 Lys Ala Asn Lys Ala Met Ile Asn Ile Asn Ile Phe Met Arg Glu
Ser785 790 795 800 Ser Arg Ser Phe Leu Val Asn Gln Met Ile Asn Glu
Ala Lys Lys Gln 805 810 815 Leu Leu Glu Phe Asp Thr Gln Ser Lys Asn
Ile Leu Met Gln Tyr Ile 820 825 830 Lys Ala Asn Ser Lys Phe Ile Gly
Ile Thr Glu Leu Lys Lys Leu Glu 835 840 845 Ser Lys Ile Asn Lys Val
Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser 850 855 860 Lys Asn Leu Asp
Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile865 870 875 880 Leu
Lys Lys Ser Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile 885 890
895 Ser Asp Ile Ser Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro Asp Ala
900 905 910 Gln Leu Val Pro Gly Ile Asn Gly Lys Ala Ile His Leu Val
Asn Asn 915 920 925 Glu Ser Ser Glu Val Ile Val His Lys Ala Met Asp
Ile Glu Tyr Asn 930 935 940 Asp Met Phe Asn Asn Phe Thr Val Ser Phe
Trp Leu Arg Val Pro Lys945 950 955 960 Val Ser Ala Ser His Leu Glu
Gln Tyr Gly Thr Asn Glu Tyr Ser Ile 965 970 975 Ile Ser Ser Met Lys
Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser 980 985 990 Val Ser Leu
Lys Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala 995 1000 1005
Gly Glu Val Arg Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe Asn
1010 1015 1020 Ala Tyr Leu Ala Asn Lys Trp Val Phe Ile Thr Ile Thr
Asn Asp Arg1025 1030 1035 1040Leu Ser Ser Ala Asn Leu Tyr Ile Asn
Gly Val Leu Met Gly Ser Ala 1045 1050 1055 Glu Ile Thr Gly Leu Gly
Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu 1060 1065 1070 Lys Leu Asp
Arg Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys 1075 1080 1085
Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys Leu
1090 1095 1100 Tyr Thr Ser Tyr Leu Ser Ile Thr Phe Leu Arg Asp Phe
Trp Gly Asn1105 1110 1115 1120Pro Leu Arg Tyr Asp Thr Glu Tyr Tyr
Leu Ile Pro Val Ala Ser Ser 1125 1130 1135 Ser Lys Asp Val Gln Leu
Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr 1140 1145 1150 Asn Ala Pro
Ser Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg 1155 1160 1165
Leu Tyr Asn Gly Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn
1170 1175 1180 Glu Ile Asp Ser Phe Val Lys Ser Gly Asp Phe Ile Lys
Leu Tyr Val1185 1190 1195 1200Ser Tyr Asn Asn Asn Glu His Ile Val
Gly Tyr Pro Lys Asp Gly Asn 1205 1210 1215 Ala Phe Asn Asn Leu Asp
Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro 1220 1225 1230 Gly Ile Pro
Leu Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu 1235 1240 1245
Lys Thr Tyr Ser Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala Ser
1250 1255 1260 Leu Gly Leu Val Gly Thr His Asn Gly Gln Ile Gly Asn
Asp Pro Asn1265 1270 1275 1280Arg Asp Ile Leu Ile Ala Ser Asn Trp
Tyr Phe Asn His Leu Lys Asp 1285 1290 1295 Lys Ile Leu Gly Cys Asp
Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp 1300 1305 1310 Thr Asn Asp
131591268PRTClostridium baratii 9Met Pro Val Asn Ile Asn Asn Phe
Asn Tyr Asn Asp Pro Ile Asn Asn1 5 10 15 Thr Thr Ile Leu Tyr Met
Lys Met Pro Tyr Tyr Glu Asp Ser Asn Lys 20 25 30 Tyr Tyr Lys Ala
Phe Glu Ile Met Asp Asn Val Trp Ile Ile Pro Glu 35 40 45 Arg Asn
Ile Ile Gly Lys Lys Pro Ser Asp Phe Tyr Pro Pro Ile Ser 50 55 60
Leu Asp Ser Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr65
70 75 80 Asp Ala Glu Lys Asp Arg Phe Leu Lys Thr Val Ile Lys Leu
Phe Asn 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Gln Val Leu Leu
Glu Glu Ile Lys 100 105 110 Asn Gly Lys Pro Tyr Leu Gly Asn Asp His
Thr Ala Val Asn Glu Phe 115 120 125 Cys Ala Asn Asn Arg Ser Thr Ser
Val Glu Ile Lys Glu Ser Asn Gly 130 135 140 Thr Thr Asp Ser Met Leu
Leu Asn Leu Val Ile Leu Gly Pro Gly Pro145 150 155 160 Asn Ile Leu
Glu Cys Ser Thr Phe Pro Val Arg Ile Phe Pro Asn Asn 165 170 175 Ile
Ala Tyr Asp Pro Ser Glu Lys Gly Phe Gly Ser Ile Gln Leu Met 180 185
190 Ser Phe Ser Thr Glu Tyr Glu Tyr Ala Phe Asn Asp Asn Thr Asp Leu
195 200 205 Phe Ile Ala Asp Pro Ala Ile Ser Leu Ala His Glu Leu Ile
His Val 210 215 220 Leu His Gly Leu Tyr Gly Ala Lys Gly Val Thr Asn
Lys Lys Val Ile225 230 235 240 Glu Val Asp Gln Gly Ala Leu Met Ala
Ala Glu Lys Asp Ile Lys Ile 245 250 255 Glu Glu Phe Ile Thr Phe Gly
Gly Gln Asp Leu Asn Ile Ile Thr Asn 260 265 270 Ser Thr Asn Gln Lys
Ile Tyr Val Ile Leu Leu Ser Asn Tyr Thr Ala 275 280 285 Ile Ala Ser
Arg Leu Ser Gln Val Asn Arg Asn Asn Ser Ala Leu Asn 290 295 300 Thr
Thr Tyr Tyr Lys Asn Phe Phe Gln Trp Lys Tyr Gly Leu Asp Gln305 310
315 320 Asp Ser Asn Gly Asn Tyr Thr Val Asn Ile Ser Lys Phe Asn Ala
Ile 325 330 335 Tyr Lys Lys Leu Phe Ser Phe Thr Glu Cys Asp Leu Ala
Gln Lys Phe 340 345 350 Gln Val Lys Asn Arg Ser Asn Tyr Leu Phe His
Phe Lys Pro Phe Arg 355 360 365 Leu Leu Asp Leu Leu Asp Asp Asn Ile
Tyr Ser Ile Ser Glu Gly Phe 370 375 380 Asn Ile Gly Ser Leu Arg Val
Asn Asn Asn Gly Gln Asn Ile Asn Leu385 390 395 400 Asn Ser Arg Ile
Val Gly Pro Ile Pro Asp Asn Gly Leu Val Glu Arg 405 410 415 Phe Val
Gly Leu Cys Lys Ser Ile Val Ser Lys Lys Gly Thr Lys Asn 420 425 430
Ser Leu Cys Ile Lys Val Asn Asn Arg Asp Leu Phe Phe Val Ala Ser 435
440 445 Glu Ser Ser Tyr Asn Glu Asn Gly Ile Asn Ser Pro Lys Glu Ile
Asp 450 455 460 Asp Thr Thr Ile Thr Asn Asn Asn Tyr Lys Lys Asn Leu
Asp Glu Val465 470 475 480 Ile Leu Asp Tyr Asn Ser Asp Ala Ile Pro
Asn Leu Ser Ser Arg Leu 485 490 495 Leu Asn Thr Thr Ala Gln Asn Asp
Ser Tyr Val Pro Lys Tyr Asp Ser 500 505 510 Asn Gly Thr Ser Glu Ile
Lys Glu Tyr Thr Val Asp Lys Leu Asn Val 515 520 525 Phe Phe Tyr Leu
Tyr Ala Gln Lys Ala Pro Glu Gly Glu Ser Ala Ile 530 535 540 Ser Leu
Thr Ser Ser Val Asn Thr Ala Leu Leu Asp Ala Ser Lys Val545 550 555
560 Tyr Thr Phe Phe Ser Ser Asp Phe Ile Asn Thr Val Asn Lys Pro Val
565 570 575 Gln Ala Ala Leu Phe Ile Ser Trp Ile Gln Gln Val Ile Asn
Asp Phe 580 585 590 Thr Thr Glu Ala Thr Gln Lys Ser Thr Ile Asp Lys
Ile Ala Asp Ile 595 600 605 Ser Leu Ile Val Pro Tyr Val Gly Leu Ala
Leu Asn Ile Gly Asn Glu 610 615 620 Val Gln Lys Gly Asn Phe Lys Glu
Ala Ile Glu Leu Leu Gly Ala Gly625 630 635 640 Ile Leu Leu Glu Phe
Val Pro Glu Leu Leu Ile Pro Thr Ile Leu Val 645 650 655 Phe Thr Ile
Lys Ser Phe Ile Asn Ser Asp Asp Ser Lys Asn Lys Ile 660 665 670 Ile
Lys Ala Ile Asn Asn Ala Leu Arg Glu Arg Glu Leu Lys Trp Lys 675 680
685 Glu Val Tyr Ser Trp Ile Val Ser Asn Trp Leu Thr Arg Ile Asn Thr
690 695 700 Gln Phe Asn Lys Arg Lys Glu Gln Met Tyr Gln Ala Leu Gln
Asn Gln705 710 715 720 Val Asp Gly Ile Lys Lys Ile Ile Glu Tyr Lys
Tyr Asn Asn Tyr Thr 725 730 735 Leu Asp Glu Lys Asn Arg Leu Arg Ala
Glu Tyr Asn Ile Tyr Ser Ile 740 745 750 Lys Glu Glu Leu Asn Lys Lys
Val Ser Leu Ala Met Gln Asn Ile Asp 755 760 765 Arg Phe Leu Thr Glu
Ser Ser Ile Ser Tyr Leu Met Lys Leu Ile Asn 770 775 780 Glu Ala Lys
Ile Asn Lys Leu Ser Glu Tyr Asp Lys Arg Val Asn Gln785 790 795 800
Tyr Leu Leu Asn Tyr Ile Leu Glu Asn Ser Ser Thr Leu Gly Thr Ser 805
810 815 Ser Val Pro Glu Leu Asn Asn Leu Val Ser Asn Thr Leu Asn Asn
Ser 820 825 830 Ile Pro Phe Glu Leu Ser Glu Tyr Thr Asn Asp Lys Ile
Leu Ile His 835 840 845 Ile Leu Ile Arg Phe Tyr Lys Arg Ile Ile Asp
Ser Ser Ile Leu Asn 850 855 860 Met Lys Tyr Glu Asn Asn Arg Phe Ile
Asp Ser Ser Gly Tyr Gly Ser865 870 875 880 Asn Ile Ser Ile Asn Gly
Asp Ile Tyr Ile Tyr Ser Thr Asn Arg Asn 885 890 895 Gln Phe Gly Ile
Tyr Ser Ser Arg Leu Ser Glu Val Asn Ile Thr Gln 900 905 910 Asn Asn
Thr Ile Ile Tyr Asn Ser Arg Tyr Gln Asn Phe Ser Val Ser 915 920 925
Phe Trp Val Arg Ile Pro Lys Tyr Asn Asn Leu Lys Asn Leu Asn Asn 930
935 940 Glu Tyr Thr Ile Ile Asn Cys Met Arg Asn Asn Asn Ser Gly Trp
Lys945 950 955 960 Ile Ser Leu Asn Tyr Asn Asn Ile Ile Trp Thr Leu
Gln Asp Thr Thr 965 970 975 Gly Asn Asn Gln Lys Leu Val Phe Asn Tyr
Thr Gln Met Ile Asp Ile 980 985 990 Ser Asp Tyr Ile Asn Lys Trp Thr
Phe Val Thr Ile Thr Asn Asn Arg 995 1000 1005 Leu Gly His Ser Lys
Leu Tyr Ile Asn Gly Asn Leu Thr Asp Gln Lys 1010 1015 1020 Ser Ile
Leu Asn Leu Gly Asn Ile His Val Asp Asp Asn Ile Leu Phe1025 1030
1035 1040Lys Ile Val Gly Cys Asn Asp Thr Arg Tyr Val Gly Ile Arg
Tyr Phe 1045 1050 1055 Lys Ile Phe Asn Met Glu Leu Asp Lys Thr Glu
Ile Glu Thr Leu Tyr 1060 1065 1070 His Ser Glu Pro Asp Ser Thr Ile
Leu Lys Asp Phe Trp Gly Asn Tyr 1075 1080 1085 Leu Leu Tyr Asn Lys
Lys Tyr Tyr Leu Leu Asn Leu Leu Lys Pro Asn 1090 1095 1100 Met Ser
Val Thr Lys Asn Ser Asp Ile Leu Asn Ile Asn Arg Gln Arg1105 1110
1115 1120Gly Ile Tyr Ser Lys Thr Asn Ile Phe Ser Asn Ala Arg Leu
Tyr Thr 1125 1130 1135 Gly Val Glu Val Ile Ile Arg Lys Val Gly Ser
Thr Asp Thr Ser Asn 1140 1145 1150 Thr Asp Asn Phe Val Arg Lys Asn
Asp Thr Val Tyr Ile Asn Val Val 1155 1160 1165 Asp Gly Asn Ser Glu
Tyr Gln Leu Tyr Ala Asp Val Ser Thr Ser Ala 1170 1175 1180 Val Glu
Lys Thr Ile Lys Leu Arg Arg Ile Ser Asn Ser Asn Tyr Asn1185 1190
1195 1200Ser Asn Gln Met Ile Ile Met Asp Ser Ile Gly Asp Asn Cys
Thr Met 1205 1210 1215 Asn Phe Lys Thr Asn Asn Gly Asn Asp Ile Gly
Leu Leu Gly Phe His 1220 1225 1230 Leu Asn Asn Leu Val Ala Ser Ser
Trp Tyr Tyr Lys Asn Ile Arg
Asn 1235 1240 1245 Asn Thr Arg Asn Asn Gly Cys Phe Trp Ser Phe Ile
Ser Lys Glu His 1250 1255 1260 Gly Trp Gln Glu1265
101251PRTClostridium butyricum 10Met Pro Thr Ile Asn Ser Phe Asn
Tyr Asn Asp Pro Val Asn Asn Arg1 5 10 15 Thr Ile Leu Tyr Ile Lys
Pro Gly Gly Cys Gln Gln Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met
Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr
Ile Pro Gln Asp Phe Leu Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60
Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Gln Glu Lys65
70 75 80 Asp Lys Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile
Asn Asp 85 90 95 Asn Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu Ser
Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Gly
Asp Phe Ile Ile Asn Asp 115 120 125 Ala Ser Ala Val Pro Ile Gln Phe
Ser Asn Gly Ser Gln Ser Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile
Met Gly Ala Glu Pro Asp Leu Phe Glu Thr145 150 155 160 Asn Ser Ser
Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Gly
Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185
190 Arg Phe Lys Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu
195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr
Gly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys
Gln Asn Pro Leu225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile
Glu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile
Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala
Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Val
Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300 Ala
Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305 310
315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr
Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln
Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu
Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile
Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu
Asn Pro Arg Ile Ile Thr Pro Ile Thr385 390 395 400 Gly Arg Gly Leu
Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 Ser Val
Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430
Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435
440 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn
Tyr 450 455 460 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser
Glu Ser Ala465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu
Thr Ile Gln Asn Asp Ala 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn
Gly Thr Ser Asp Ile Glu Gln His 500 505 510 Asp Val Asn Glu Leu Asn
Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu
Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu
Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile545 550 555
560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Gly Trp Ile
565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys
Ser Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro
Tyr Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys
Gly Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile
Leu Leu Glu Phe Glu Pro Glu Leu625 630 635 640 Leu Ile Pro Thr Ile
Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn
Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu
Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680
685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met
690 695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Leu Lys Ala Ile
Ile Glu705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys
Asn Glu Leu Thr Asn 725 730 735 Lys Tyr Asp Ile Glu Gln Ile Glu Asn
Glu Leu Asn Gln Lys Val Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp
Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu
Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu
Asn Val Lys Thr Tyr Leu Leu Asp Tyr Ile Ile Lys His785 790 795 800
Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Ile 805
810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr
Asp 820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys
Arg Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn
Asp Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn
Ile Asn Gly Asp Val Tyr Lys865 870 875 880 Tyr Pro Thr Asn Lys Asn
Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile
Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn
Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925
Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930
935 940 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile
Ile945 950 955 960 Trp Thr Leu Gln Asp Asn Ser Gly Ile Asn Gln Lys
Leu Ala Phe Asn 965 970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr
Ile Asn Lys Trp Ile Phe 980 985 990 Val Thr Ile Thr Asn Asp Arg Leu
Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp
Lys Lys Ser Ile Leu Asn Leu Gly Asn Ile His 1010 1015 1020 Val Ser
Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr Thr Arg1025 1030
1035 1040Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu Leu
Asp Glu 1045 1050 1055 Thr Glu Ile Gln Thr Leu Tyr Asn Asn Glu Pro
Asn Ala Asn Ile Leu 1060 1065 1070 Lys Asp Phe Trp Gly Asn Tyr Leu
Leu Tyr Asp Lys Glu Tyr Tyr Leu 1075 1080 1085 Leu Asn Val Leu Lys
Pro Asn Asn Phe Ile Asn Arg Arg Thr Asp Ser 1090 1095 1100 Thr Leu
Ser Ile Asn Asn Ile Arg Ser Thr Ile Leu Leu Ala Asn Arg1105 1110
1115 1120Leu Tyr Ser Gly Ile Lys Val Lys Ile Gln Arg Val Asn Asn
Ser Ser 1125 1130 1135 Thr Asn Asp Asn Leu Val Arg Lys Asn Asp Gln
Val Tyr Ile Asn Phe 1140 1145 1150 Val Ala Ser Lys Thr His Leu Leu
Pro Leu Tyr Ala Asp Thr Ala Thr 1155 1160 1165 Thr Asn Lys Glu Lys
Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe 1170 1175 1180 Asn Gln
Val Val Val Met Asn Ser Val Gly Asn Cys Thr Met Asn Phe1185 1190
1195 1200Lys Asn Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys
Ala Asp 1205 1210 1215 Thr Val Val Ala Ser Thr Trp Tyr Tyr Thr His
Met Arg Asp Asn Thr 1220 1225 1230 Asn Ser Asn Gly Phe Phe Trp Asn
Phe Ile Ser Glu Glu His Gly Trp 1235 1240 1245 Gln Glu Lys 1250
1125PRTArtificial SequenceBoNT/A di-chain loop region 11Cys Val Arg
Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly1 5 10 15 Tyr
Asn Lys Ala Leu Asn Asp Leu Cys 20 25 1210PRTArtificial
SequenceBoNT/B di-chain loop region 12Cys Lys Ser Val Lys Ala Pro
Gly Ile Cys1 5 10 1317PRTArtificial SequenceBoNT/C1 di-chain loop
region 13Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn Lys Thr
Leu Asp1 5 10 15 Cys1414PRTArtificial SequenceBoNT/D di-chain loop
region 14Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser Thr Cys1 5
10 1515PRTArtificial SequenceBoNT/E di-chain loop region 15Cys Lys
Asn Ile Val Ser Val Lys Gly Ile Arg Lys Ser Ile Cys1 5 10 15
1617PRTArtificial SequenceBoNT/F di-chain loop region 16Cys Lys Ser
Val Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu1 5 10 15
Cys1715PRTArtificial SequenceBoNT/G di-chain loop region 17Cys Lys
Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu Gln Cys1 5 10 15
1829PRTArtificial SequenceTeNT di-chain loop region 18Cys Lys Lys
Ile Ile Pro Pro Thr Asn Ile Arg Glu Asn Leu Tyr Asn1 5 10 15 Arg
Thr Ala Ser Leu Thr Asp Leu Gly Gly Glu Leu Cys 20 25
1915PRTArtificial SequenceBaNT di-chain loop region 19Cys Lys Ser
Ile Val Ser Lys Lys Gly Thr Lys Asn Ser Leu Cys1 5 10 15
2015PRTArtificial SequenceBuNT di-chain loop region 20Cys Lys Asn
Ile Val Ser Val Lys Gly Ile Arg Lys Ser Ile Cys1 5 10 15
215PRTArtificial SequenceG-spacer 21Gly Gly Gly Gly Ser1 5
225PRTArtificial SequenceA-spacer 22Glu Ala Ala Ala Lys1 5
238PRTArtificial SequenceFLAG epitope-binding region 23Asp Tyr Lys
Asp Asp Asp Asp Lys1 5 249PRTArtificial SequenceHuman Influenza
virus hemagluttinin (HA) epitope-binding region 24Tyr Pro Tyr Asp
Val Pro Asp Tyr Ala1 5 2510PRTArtificial SequenceHuman p62 c-Myc
epitope-binding region 25Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu1 5
10 2611PRTArtificial SequenceVesicular Stomatitis Virus
Glycoprotein (VSV-G) epitope-binding region 26Tyr Thr Asp Ile Glu
Met Asn Arg Leu Gly Lys1 5 10 276PRTArtificial SequenceSubstance P
epitope-binding region 27Gln Phe Phe Gly Leu Met1 5
2811PRTArtificial SequenceGlycoprotein-D precursor of Herpes
simplex virus epitope-binding region 28Gln Pro Glu Leu Ala Pro Glu
Asp Pro Glu Asp1 5 10 2914PRTArtificial SequenceV5 epitope-binding
region 29Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr1 5
10 306PRTArtificial SequenceAU1 epitope-binding region 30Asp Thr
Tyr Arg Tyr Ile1 5 316PRTArtificial SequenceAU5 epitope-binding
region 31Thr Asp Phe Tyr Leu Lys1 5 326PRTArtificial SequenceHIS
epitope-binding region 32His His His His His His1 5
33527PRTClostridium botulinum serotype A 33Met Tyr Ile Met Leu Lys
Asn Lys Leu Ser Ile Leu Leu Thr Ser Thr1 5 10 15 Leu Val Ala Ala
Ser Leu Leu Ser Phe Lys Pro Val Tyr Ala Asp Ser 20 25 30 Val Asn
Asn Ser Asn Lys Thr Asn Leu Asn Asn Val Arg Gln Glu Ile 35 40 45
Gln Lys Ala Lys Asn Asn Asn Gln Lys Val Thr Ile Met Tyr Tyr Cys 50
55 60 Asp Ala Asp Asn Asn Leu Glu Ser Ser Leu Leu Ser Asp Ile Glu
Glu65 70 75 80 Met Lys Lys Gly Tyr Val Asn Asn Pro Asn Leu Asn Leu
Val Thr Leu 85 90 95 Ile Asp Arg Ser Ser Arg Tyr Thr Ser Asp Lys
Thr Val Phe Gly Glu 100 105 110 Asp Phe Glu Asp Ala Arg Leu Tyr Lys
Ile Glu His Asn Lys Thr Lys 115 120 125 Arg Leu Asp Gly Gly Lys Glu
Phe Pro Glu Ile Thr Leu Asn Ser Asn 130 135 140 Tyr Glu Ala Asn Met
Gly Asp Ala Asp Thr Leu Lys Lys Phe Ile Asn145 150 155 160 Tyr Cys
Lys Ala Asn Tyr Lys Ala Asp Lys Tyr Val Leu Ile Met Ala 165 170 175
Asn His Gly Gly Gly Ala Lys Glu Lys Leu Lys Asn Asn Gln Asp Val 180
185 190 Asn Arg Ala Ile Cys Trp Asp Asp Ser His Tyr Asp Gly Asn Ser
Pro 195 200 205 Asp Cys Leu Tyr Met Gly Glu Ile Ser Asp Thr Leu Thr
Gln Glu Gln 210 215 220 Ser Val Asp Val Leu Ala Phe Asp Ala Cys Leu
Met Gly Thr Ala Glu225 230 235 240 Val Ala Tyr Gln Phe Arg Pro Gly
Asn Gly Gly Phe Ser Ala Asp Gly 245 250 255 Ile Val Ala Ser Ser Pro
Val Val Trp Gly Pro Gly Phe Gln Tyr Asp 260 265 270 Asn Ile Leu Ser
Arg Leu Lys Ser Gly Gly Gly Ser Ser Asn Glu Asp 275 280 285 Asp Leu
Thr Leu Gly Gly Lys Glu Lys Asn Phe Asp Pro Ala Thr Ile 290 295 300
Thr Asn Glu Gln Leu Gly Ala Leu Phe Val Glu Glu Gln Arg Asp Ser305
310 315 320 Thr His Ala Arg Gly Ser Tyr Asp Gln His Leu Ser Phe Tyr
Asp Ala 325 330 335 Ala Lys Val Glu Asp Val Lys Arg Ser Ile Asp Asn
Leu Ala Ile Asn 340 345 350 Leu Ser Asn Glu Asn Lys Lys Asp Gly Ile
Glu Lys Leu Arg Gly Ser 355 360 365 Lys Asn Asn Thr Asn Leu Met His
Tyr Phe Asp Glu Tyr Asn Glu Gln 370 375 380 Glu Trp Ile Glu Tyr Pro
Tyr Phe Asp Ile Tyr Asp Leu Cys Glu Lys385 390 395 400 Ile Asn Glu
Ser Asn Asn Phe Ser Asn Glu Thr Arg Thr Leu Ala Ser 405 410 415 Ile
Cys Met Asp Lys Ile Asp Glu Met Val Val Tyr Ser Phe Gly Gly 420 425
430 Pro Ser Lys Glu Phe Lys Glu Gly Lys Asn Gly Leu Ser Ile Phe Leu
435 440 445 Pro Asp Gly Asp Lys Lys Tyr Ser Asn Tyr Tyr Asn Ser Ile
Pro His 450 455 460 Trp Thr Ile Gln Ser Trp Tyr Asn Ser Ile Asp Thr
Val Gln Asn Gly465 470 475 480 Leu Pro Pro Tyr Gly Lys Leu Thr Trp
Cys Gln Asp Gly Gln Asp Pro 485 490 495 Arg Val Asn Lys Val Gly Asn
Trp Phe Glu Leu Leu Asp Ser Trp Phe 500 505 510 Asp Lys Thr Asn Gly
Ala Asp Gly Gly Val Asn His Tyr Gln Trp 515 520 525
34526PRTClostridium histolyticum 34 Met Leu Arg Arg Lys Val Ser Thr
Leu Leu Met Thr Ala Leu Ile Thr1 5 10 15 Thr Ser Phe Leu Asn Ser
Lys Pro Val Tyr Ala Asn Pro Val Thr Lys 20 25 30 Ser Lys Asp Asn
Asn Leu Lys Glu Val Gln Gln Val Thr Ser Lys Ser 35 40 45 Asn
Lys
Asn Lys Asn Gln Lys Val Thr Ile Met Tyr Tyr Cys Asp Ala 50 55 60
Asp Asn Asn Leu Glu Gly Ser Leu Leu Asn Asp Ile Glu Glu Met Lys65
70 75 80 Thr Gly Tyr Lys Asp Ser Pro Asn Leu Asn Leu Ile Ala Leu
Val Asp 85 90 95 Arg Ser Pro Arg Tyr Ser Ser Asp Glu Lys Val Leu
Gly Glu Asp Phe 100 105 110 Ser Asp Thr Arg Leu Tyr Lys Ile Glu His
Asn Lys Ala Asn Arg Leu 115 120 125 Asp Gly Lys Asn Glu Phe Pro Glu
Ile Ser Thr Thr Ser Lys Tyr Glu 130 135 140 Ala Asn Met Gly Asp Pro
Glu Val Leu Lys Lys Phe Ile Asp Tyr Cys145 150 155 160 Lys Ser Asn
Tyr Glu Ala Asp Lys Tyr Val Leu Ile Met Ala Asn His 165 170 175 Gly
Gly Gly Ala Arg Glu Lys Ser Asn Pro Arg Leu Asn Arg Ala Ile 180 185
190 Cys Trp Asp Asp Ser Asn Leu Asp Lys Asn Gly Glu Ala Asp Cys Leu
195 200 205 Tyr Met Gly Glu Ile Ser Asp His Leu Thr Glu Lys Gln Ser
Val Asp 210 215 220 Leu Leu Ala Phe Asp Ala Cys Leu Met Gly Thr Ala
Glu Val Ala Tyr225 230 235 240 Gln Tyr Arg Pro Gly Asn Gly Gly Phe
Ser Ala Asp Thr Leu Val Ala 245 250 255 Ser Ser Pro Val Val Trp Gly
Pro Gly Phe Lys Tyr Asp Lys Ile Phe 260 265 270 Asp Arg Ile Lys Ala
Gly Gly Gly Thr Asn Asn Glu Asp Asp Leu Thr 275 280 285 Leu Gly Gly
Lys Glu Gln Asn Phe Asp Pro Ala Thr Ile Thr Asn Glu 290 295 300 Gln
Leu Gly Ala Leu Phe Val Glu Glu Gln Arg Asp Ser Thr His Ala305 310
315 320 Asn Gly Arg Tyr Asp Gln His Leu Ser Phe Tyr Asp Leu Lys Lys
Ala 325 330 335 Glu Ser Val Lys Arg Ala Ile Asp Asn Leu Ala Val Asn
Leu Ser Asn 340 345 350 Glu Asn Lys Lys Ser Glu Ile Glu Lys Leu Arg
Gly Ser Gly Ile His 355 360 365 Thr Asp Leu Met His Tyr Phe Asp Glu
Tyr Ser Glu Gly Glu Trp Val 370 375 380 Glu Tyr Pro Tyr Phe Asp Val
Tyr Asp Leu Cys Glu Lys Ile Asn Lys385 390 395 400 Ser Glu Asn Phe
Ser Ser Lys Thr Lys Asp Leu Ala Ser Asn Ala Met 405 410 415 Asn Lys
Leu Asn Glu Met Ile Val Tyr Ser Phe Gly Asp Pro Ser Asn 420 425 430
Asn Phe Lys Glu Gly Lys Asn Gly Leu Ser Ile Phe Leu Pro Asn Gly 435
440 445 Asp Lys Lys Tyr Ser Thr Tyr Tyr Thr Ser Thr Lys Ile Pro His
Trp 450 455 460 Thr Met Gln Ser Trp Tyr Asn Ser Ile Asp Thr Val Lys
Tyr Gly Leu465 470 475 480 Asn Pro Tyr Gly Lys Leu Ser Trp Cys Lys
Asp Gly Gln Asp Pro Glu 485 490 495 Ile Asn Lys Val Gly Asn Trp Phe
Glu Leu Leu Asp Ser Trp Phe Asp 500 505 510 Lys Thr Asn Asp Val Thr
Gly Gly Val Asn His Tyr Gln Trp 515 520 525 35530PRTClostridium
novyi 35Met Leu Arg Lys Lys Val Ile His Leu Leu Ala Ala Ala Ile Leu
Gly1 5 10 15 Met Ser Phe Ile Asn Ile Gln Pro Val Tyr Ala Thr Pro
Lys Pro Thr 20 25 30 Ala Asn Asn Ala Lys Ser Ser Ile Thr Lys Lys
Ala Lys Asp Glu Ser 35 40 45 Lys Lys Glu Gln Lys Val Thr Ile Leu
Tyr Tyr Cys Asp Ala Asp Asn 50 55 60 Asn Leu Glu Asp Ala Leu Met
Ser Asp Ile Ala Glu Met Lys Lys Gly65 70 75 80 Tyr Val Asn Asn Pro
Asn Leu Asn Leu Ile Ala Leu Val Asp Arg Thr 85 90 95 Pro Gly Tyr
Ser Glu Asp Ser Thr Ala Leu Gly Glu Asn Phe Glu Asp 100 105 110 Thr
Arg Leu Tyr Lys Ile Glu His Asn Lys Ala Thr Arg Leu Asp Gly 115 120
125 Gly Lys Tyr Phe Pro Glu Ile Lys Val Asn Gly Thr Tyr Glu Ala Asn
130 135 140 Met Gly Asp Pro Glu Thr Leu Lys Lys Phe Ile Glu Phe Gly
Lys Asn145 150 155 160 Asn Tyr Lys Ala Asp Lys Tyr Val Leu Ile Met
Ser Asn His Gly Gly 165 170 175 Gly Ala Lys Asn Lys Pro Asn Val Asn
Glu Lys Leu Asn Lys Ala Ile 180 185 190 Cys Trp Asp Asp Ser Asn Leu
Asp Gly Glu Asn Pro Asp Cys Leu Tyr 195 200 205 Ile Gly Glu Ile Ser
Asp His Leu Asp Glu Ser His Ser Val Asp Val 210 215 220 Leu Ala Phe
Asp Ala Cys Leu Met Gly Thr Ala Glu Val Ala Tyr Gln225 230 235 240
Tyr Arg Pro Gly Asn Gly Arg Phe Ser Ala Lys Thr Met Ile Ala Ser 245
250 255 Ser Pro Val Val Trp Gly Ala Gly Phe Lys Tyr Asp Asp Ile Phe
Ser 260 265 270 Arg Ile Arg Ser Gly Asn Thr Phe Thr Leu Gln Lys Asp
Leu Thr Leu 275 280 285 Gly Gly Arg Glu Arg Cys Phe Asp Pro Ala Thr
Ile Thr Asn Glu Gln 290 295 300 Ile Gly Ala Leu Phe Val Glu Glu Gln
Arg Asp Ser Thr Ser Arg Tyr305 310 315 320 Phe Arg Ser Asp Gln Gln
Leu Ser Cys Tyr Asp Leu Thr Lys Ala Glu 325 330 335 Asn Ile Lys Lys
Ser Phe Asp Lys Leu Ala Val Asp Leu Ser Thr Lys 340 345 350 Asn Lys
Lys Ser Ala Ile Glu Lys Leu Arg Gly Ser Lys Thr Asn Val 355 360 365
Asn Leu Met His Tyr Phe Asn Glu Lys Asp Gln Leu Asp Trp Ile Glu 370
375 380 Tyr Pro Tyr Phe Asp Ile Tyr Asp Leu Cys Glu Gly Ile Ser Lys
Ser385 390 395 400 Asn Asp Phe Asp Glu Glu Thr Gln Lys Leu Ala Lys
Asp Val Met Lys 405 410 415 Asn Val Asp Ser Met Val Leu Tyr Ser Phe
Gly Gly Lys Lys Phe Lys 420 425 430 Gly Val Gly Lys Phe Lys Glu Gly
Lys Asn Gly Leu Ser Val Phe Leu 435 440 445 Pro Asp Gly Asn Arg Ile
Tyr Thr Ser Arg Tyr Ser Asn Ala Lys Ile 450 455 460 Pro His Trp Leu
Ile Gln Ser Trp Tyr Asn Ser Ile Asp Thr Val Ala465 470 475 480 Ser
Gly Leu Asn Asn Pro Tyr Gly Lys Leu Ser Trp Cys Lys Asp Gly 485 490
495 Gln Asp Pro Glu Ile Asn Lys Val Gly Asn Trp Phe Glu Leu Leu Asp
500 505 510 Ser Trp Phe Asp Lys Thr Asn Gly Pro Asp Gly Gly Phe Asn
His Tyr 515 520 525 Gln Trp 530 36522PRTClostridium perfringens
36Met Phe Lys Lys Lys Leu Ser Leu Leu Met Ala Thr Ile Thr Ile Gly1
5 10 15 Ser Val Leu Leu Gly Gly Ala Ser Thr Val Ser Ala Ala Pro Arg
Gln 20 25 30 Lys His Lys Thr Val Ser Glu Lys Ile Lys Glu Ala Glu
Lys Thr Glu 35 40 45 Gly Asp Lys Lys Leu Thr Val Met Val Tyr Ala
Asp Cys Asp Asn Asn 50 55 60 Leu Glu Glu Tyr Ile Leu Asn Asp Ile
Glu Glu Met Lys Glu Gly Tyr65 70 75 80 Lys Asn Asn Pro Asn Leu Asn
Ile Val Val Leu Val Asp Arg Ile Pro 85 90 95 Gly Tyr Ser Asn Asp
Ser Lys Val Leu Gly Ser Asn Phe Glu Asp Thr 100 105 110 Arg Leu Tyr
Lys Ile Gly Glu Asn Ser Ala Glu Arg Ile Ser Gly Lys 115 120 125 Ser
Glu Phe Pro Glu Ile Thr Thr Thr Ser Asn Tyr Glu Ala Asn Met 130 135
140 Gly Asp Ala Asn Thr Leu Lys Lys Phe Ile Lys Phe Cys Lys Lys
Asn145 150 155 160 Tyr Glu Ala Asp Lys Tyr Met Leu Ile Met Ser Asn
His Gly Gly Gly 165 170 175 Ala Lys Asp Asp Lys Asp Arg Ala Ser Thr
Val Asn Lys Ala Ile Cys 180 185 190 Trp Asp Asp Ser Asn Asn Lys Asp
Cys Leu Tyr Thr Gly Glu Ile Ser 195 200 205 Asp Val Leu Thr Lys Asp
Glu Ser Val Asp Val Leu Val Phe Asp Ala 210 215 220 Cys Leu Met Gly
Thr Ser Glu Val Ala Tyr Gln Tyr Arg Pro Asn Asn225 230 235 240 Gly
Ser Phe Glu Ala Lys Thr Leu Val Ala Ser Ala Pro Val Val Trp 245 250
255 Gly Asn Gly Tyr Pro Tyr Asp Lys Ile Phe Ser Arg Leu Lys Ser Thr
260 265 270 Lys Gly Asp Asn Gly Glu Val Asp Ser Thr Leu Gly Gly Lys
Glu Lys 275 280 285 Ile Phe Glu Pro Ser Leu Val Thr Asn Asn Glu Leu
Gly Ala Leu Phe 290 295 300 Val Glu Glu Gln Arg Asp Ser Val Asn Ser
Tyr Gly Val Thr Asp Gln305 310 315 320 Gln Leu Ser Cys Tyr Asp Leu
Ser Lys Ile Glu Thr Val Lys Lys Ser 325 330 335 Val Asp Ala Leu Ala
Arg Asn Leu Ser Lys Asn Asn Lys Lys Asp Ala 340 345 350 Ile Glu Asn
Leu Arg Gly Thr Gly Lys Asn Ala Pro Thr Met His Tyr 355 360 365 Phe
Lys Asn Tyr Asp Glu Tyr Glu Trp Ile Glu Tyr Pro Tyr Phe Asp 370 375
380 Leu Tyr Asp Leu Cys Glu Lys Ile Ser Leu Ser Asn Glu Phe Asp
Glu385 390 395 400 Thr Thr Lys Lys Leu Ser Lys Asn Val Met Lys Asn
Val Asp Gln Leu 405 410 415 Ile Leu Tyr Ser Phe Ala Gly Asn Asp Phe
Lys Gly Phe Lys Glu Gly 420 425 430 Lys Asn Gly Ile Ser Ile Phe Leu
Pro Asp Gly Asn Arg Asn Tyr Tyr 435 440 445 Asp Gln Tyr Ser Gly Gln
Val Ile Pro His Trp Ala Ile Gln Arg Trp 450 455 460 Tyr Asn Pro Leu
Asp Thr Asn Ala Tyr Arg Leu Arg Ser Gly Tyr Gly465 470 475 480 Lys
Leu Ala Trp Cys Lys Asp Gly Leu Asp Pro Lys Ile Asn Lys Val 485 490
495 Gly Asn Trp Phe Glu Leu Leu Asp Ser Trp Phe Asp Lys Asp Asn Thr
500 505 510 Ser Leu Gly Gly Tyr Asn Arg Tyr Arg Tyr 515 520
37522PRTClostridium perfringens 37Met Phe Lys Lys Lys Leu Ser Leu
Leu Met Ala Thr Ile Thr Ile Gly1 5 10 15 Ser Val Leu Leu Gly Gly
Val Ser Thr Val Ser Ala Ala Pro Arg Gln 20 25 30 Lys His Lys Thr
Val Ser Glu Lys Ile Lys Glu Ala Glu Lys Thr Glu 35 40 45 Gly Asp
Lys Lys Leu Thr Val Met Val Tyr Ala Asp Cys Asp Asn Asn 50 55 60
Leu Glu Glu Tyr Ile Leu Asn Asp Ile Glu Glu Met Lys Glu Gly Tyr65
70 75 80 Lys Asn Asn Pro Asn Leu Asn Ile Ile Val Leu Val Asp Arg
Ile Pro 85 90 95 Gly Tyr Ser Asn Asp Ser Lys Val Leu Gly Ser Asn
Phe Glu Asp Thr 100 105 110 Arg Leu Tyr Lys Ile Gly Glu Asn Ser Ala
Glu Arg Ile Ser Gly Lys 115 120 125 Ser Glu Phe Pro Glu Ile Thr Thr
Thr Ser Asn Tyr Glu Ala Asn Met 130 135 140 Gly Asp Ala Asn Thr Leu
Lys Lys Phe Ile Lys Phe Cys Lys Lys Asn145 150 155 160 Tyr Glu Ala
Asp Lys Tyr Met Leu Ile Met Ser Asn His Gly Gly Gly 165 170 175 Ala
Lys Asp Asp Lys Asp Arg Ala Ser Thr Val Asn Lys Ala Ile Cys 180 185
190 Trp Asp Asp Ser Asn Asn Lys Asp Cys Leu Tyr Thr Gly Glu Ile Ser
195 200 205 Asp Val Leu Thr Lys Asp Glu Ser Val Asp Val Leu Val Phe
Asp Ala 210 215 220 Cys Leu Met Gly Asn Ser Glu Val Ala Tyr Gln Tyr
Arg Pro Asn Asn225 230 235 240 Gly Ser Phe Glu Ala Lys Thr Leu Val
Ala Ser Ala Pro Val Val Trp 245 250 255 Gly Phe Gly Tyr Pro Tyr Asp
Lys Ile Phe Ser Arg Leu Arg Ser Thr 260 265 270 Lys Gly Asp Asn Gly
Glu Val Asp Ser Thr Leu Gly Gly Lys Glu Lys 275 280 285 Ile Phe Asp
Pro Ser Thr Val Thr Asn Asn Glu Leu Gly Ala Leu Phe 290 295 300 Val
Glu Glu Gln Arg Asp Ser Val Asn Ser Cys Gly Val Thr Asp Gln305 310
315 320 Gln Leu Ser Cys Tyr Asp Leu Ser Lys Ile Glu Lys Val Lys Lys
Ser 325 330 335 Val Asp Thr Leu Ala Arg Asn Leu Ser Lys Asn Asn Lys
Lys Asp Ala 340 345 350 Ile Glu Ser Leu Arg Gly Thr Gly Lys Asn Ala
Pro Thr Met His Tyr 355 360 365 Phe Lys Asn Tyr Asp Glu Tyr Glu Trp
Ile Glu Tyr Pro Tyr Phe Asp 370 375 380 Leu Tyr Asp Leu Cys Glu Lys
Ile Ser Leu Ser Asp Glu Phe Asn Glu385 390 395 400 Thr Thr Lys Lys
Leu Ser Lys Asn Val Met Lys Asn Val Asp Gln Leu 405 410 415 Ile Leu
Tyr Ser Phe Ala Gly Asn Asp Phe Lys Gly Phe Lys Glu Gly 420 425 430
Lys Asn Gly Ile Ser Ile Phe Leu Pro Asp Gly Asn Arg Asn Tyr Tyr 435
440 445 Asp Gln Tyr Ser Gly Gln Ala Ile Pro His Trp Ala Ile Gln Arg
Trp 450 455 460 Tyr Asn Pro Leu Asp Thr Asn Ala Tyr Arg Leu Arg Ser
Gly Tyr Gly465 470 475 480 Lys Leu Ser Trp Cys Lys Asp Gly Leu Asp
Pro Lys Ile Asn Lys Val 485 490 495 Gly Asn Trp Phe Glu Leu Leu Asp
Ser Trp Phe Asp Lys Asp Asn Thr 500 505 510 Ser Leu Gly Gly Tyr Asn
Arg Tyr Arg Tyr 515 520 38524PRTClostridium perfringens 38 Met Tyr
Met Phe Lys Lys Lys Leu Ser Leu Leu Met Ala Thr Ile Thr1 5 10 15
Ile Gly Ser Val Leu Leu Gly Gly Val Ser Thr Val Ser Ala Ala Pro 20
25 30 Arg Gln Lys His Lys Thr Val Ser Glu Lys Ile Lys Glu Ala Glu
Lys 35 40 45 Thr Glu Gly Asp Lys Lys Leu Thr Val Met Val Tyr Ala
Asp Cys Asp 50 55 60 Asn Asn Leu Glu Glu Tyr Ile Leu Asn Asp Ile
Glu Glu Met Lys Glu65 70 75 80 Gly Tyr Lys Asn Asn Pro Asn Leu Asn
Ile Ile Val Leu Val Asp Arg 85 90 95 Ile Pro Gly Tyr Ser Asn Asp
Ser Lys Val Leu Gly Ser Asn Phe Glu 100 105 110 Asp Thr Arg Leu Tyr
Lys Ile Gly Glu Asn Ser Ala Glu Arg Ile Ser 115 120 125 Gly Lys Ser
Glu Phe Pro Glu Ile Thr Thr Thr Ser Asn Tyr Glu Ala 130 135 140 Asn
Met Gly Asp Ala Asn Thr Leu Lys Lys Phe Ile Lys Phe Cys Lys145 150
155 160 Lys Asn Tyr Glu Ala Asp Lys Tyr Met Leu Ile Met Ser Asn His
Gly 165 170 175 Gly Gly Ala Lys Asp Asp Lys Asp Arg Ala Ser Thr Val
Asn Lys Ala 180 185 190 Ile Cys Trp Asp Asp Ser Asn Asn Lys Asp Cys
Leu Tyr Thr Gly Glu 195 200 205 Ile Ser Asp Val Leu Thr Lys Asp Glu
Ser Val Asp Val Leu Val Phe 210 215 220 Asp Ala Cys Leu Met Gly Asn
Ser Glu Val Ala Tyr Gln Tyr Arg Pro225 230 235 240 Asn Asn Gly Ser
Phe
Glu Ala Lys Thr Leu Val Ala Ser Ala Pro Val 245 250 255 Val Trp Gly
Phe Gly Tyr Pro Tyr Asp Lys Ile Phe Ser Arg Leu Arg 260 265 270 Ser
Thr Lys Gly Asp Asn Gly Glu Val Asp Ser Thr Leu Gly Gly Lys 275 280
285 Glu Lys Ile Phe Asp Pro Ser Thr Val Thr Asn Asn Glu Leu Gly Ala
290 295 300 Leu Phe Val Glu Glu Gln Arg Asp Ser Val Asn Ser Cys Arg
Val Thr305 310 315 320 Asp Gln Gln Leu Ser Cys Tyr Asp Leu Ser Lys
Ile Glu Lys Val Lys 325 330 335 Lys Ser Val Asp Ala Leu Ala Arg Asn
Leu Ser Lys Asn Asn Lys Lys 340 345 350 Asp Ala Ile Glu Lys Leu Arg
Gly Thr Gly Lys Asn Ala Pro Thr Met 355 360 365 His Tyr Phe Lys Asn
Tyr Asp Glu Tyr Glu Trp Ile Glu Tyr Pro Tyr 370 375 380 Phe Asp Leu
Tyr Asp Leu Cys Glu Lys Ile Ser Leu Ser Asp Glu Phe385 390 395 400
Asn Glu Thr Thr Lys Lys Leu Ser Lys Asn Val Met Lys Asn Val Asp 405
410 415 Gln Leu Ile Leu Tyr Ser Phe Ala Gly Lys Asp Phe Lys Gly Phe
Lys 420 425 430 Glu Gly Lys Asn Gly Ile Ser Ile Phe Leu Pro Asp Gly
Asn Arg Asn 435 440 445 Tyr Tyr Asp Gln Tyr Ser Gly Gln Ala Ile Pro
His Trp Ala Ile Gln 450 455 460 Arg Trp Tyr Asn Pro Leu Asp Thr Asn
Ala Tyr Arg Leu Arg Ser Gly465 470 475 480 Tyr Gly Lys Leu Ala Trp
Cys Lys Asp Gly Leu Asp Pro Lys Ile Asn 485 490 495 Lys Val Gly Asn
Trp Phe Glu Leu Leu Asp Ser Trp Phe Asp Lys Asp 500 505 510 Asn Thr
Ser Leu Gly Gly Tyr Asn Arg Tyr Arg Tyr 515 520 39772PRTClostridium
botulinum serotype A 39Met Leu Arg Lys Ile Glu Ser Lys Asp Leu Leu
Tyr Asn Leu Glu Leu1 5 10 15 Asp Asp Ile Asn Ile Asp Arg Thr Gln
Lys Tyr Thr Pro Glu Tyr Asn 20 25 30 Ser Ile Tyr Glu Lys Ile Asn
Leu Ala Leu Asp Ile Asp Lys Ser Gly 35 40 45 Tyr Asn Leu Tyr Leu
Val Asp Asp Phe Ser Lys Glu Lys Leu Asn Ser 50 55 60 Ile Ile Asn
Phe Ile Asn Gln Lys Leu Glu Lys Lys Ser Lys Pro Lys65 70 75 80 Asp
Ile Cys Tyr Val Val Leu Glu Glu Glu Arg Tyr Pro Tyr Ser Ile 85 90
95 Tyr Leu Glu Asn Gly Lys Gly Lys Ile Leu Lys Glu Lys Leu Lys Asp
100 105 110 Ile Gln Thr Lys Tyr Asn Glu Cys Ile Tyr Asp Phe Tyr Asn
Lys Ser 115 120 125 Ser Asn Lys Glu Lys Glu Ile Ile Leu Glu Ser Met
Glu Lys Lys Arg 130 135 140 Ser Glu Ile Val Asn Glu Leu Ile Glu Glu
Ser Lys Lys Glu Gly Phe145 150 155 160 Glu Ile Lys Thr Gly Val Ser
Gly Phe Val Phe Met Pro Ile Lys Asp 165 170 175 Gly Asn Ser Leu Ser
Glu Ser Glu Tyr Glu Asp Leu Asn Lys Glu Asp 180 185 190 Lys Glu Glu
Ile Leu Glu Lys Val Ser Lys Leu Lys Glu Lys Ala Glu 195 200 205 Ser
Ser Leu Glu Val Leu Ala Asp Met Glu Arg Glu Gly Leu Glu Lys 210 215
220 Leu Lys Asp Ile Met Arg Thr Tyr Leu Glu Met Glu Met Lys Gly
Ser225 230 235 240 Lys Glu Glu Tyr Arg Met Glu Phe Glu Asp Asn Ile
Gln Thr Leu Asp 245 250 255 Phe Leu Asn Ser Val Cys Arg Asn Ile Glu
Lys Glu Leu Ile Glu Ser 260 265 270 Tyr Thr Ser Ser Tyr Glu Glu Asp
Gln Glu Ser Ile Ile Asn Val Ile 275 280 285 Tyr Lys Tyr Lys Val Asn
Val Ile Val Asp Asn Thr Leu Asn Lys Ser 290 295 300 Pro Leu Val Ile
Phe Glu Glu Asn Pro Ser Val Asn Asn Leu Val Gly305 310 315 320 Ser
Ile Glu Tyr Glu Asn Lys Ser Gly Val Tyr Tyr Thr Asp Ala Ser 325 330
335 Leu Ile Lys Ala Gly Ser Leu Leu Lys Ala Asn Glu Gly Cys Leu Ile
340 345 350 Val Arg Ala Asn Ser Leu Phe Thr Asn Gly Ser Ala Tyr Phe
Tyr Leu 355 360 365 Lys Lys Ala Leu Ile Asn Asp Lys Ile Asp Phe Asp
Tyr Asn Lys Gly 370 375 380 Tyr Leu Glu Leu Leu Ser Leu Gly Gly Leu
Lys Pro Glu Pro Ile Asn385 390 395 400 Thr Lys Leu Lys Val Ile Ile
Ile Gly Asp Tyr Glu Thr Tyr Asn Leu 405 410 415 Leu Tyr Asn Tyr Asp
Glu Asp Phe Lys Lys Ile Phe Lys Leu Lys Ser 420 425 430 Glu Tyr Asn
Lys Val Val Asp Ile Asn Ser Lys Ser Lys Glu Gln Ile 435 440 445 Cys
Lys Asn Ile Tyr Asp Ile Cys Glu Asn Lys Asp Leu Lys Asn Ile 450 455
460 Asn Glu Glu Ala Val Lys Glu Val Cys Lys Tyr Leu Ser Arg Lys
Ala465 470 475 480 Glu Asn Lys Asn Lys Phe Tyr Phe Asp Asn Tyr Glu
Ile Asp Arg Leu 485 490 495 Leu Ile Gln Ala Asp Ser Lys Ala Arg Ile
Glu Asp Arg Asp Ile Ile 500 505 510 Thr Lys Glu Asp Ile Gln Phe Val
Ala Tyr Glu Lys Glu Glu Ile Glu 515 520 525 Lys Glu Val Met Glu Gly
Tyr Glu Lys Glu Arg Ile Phe Ile Asp Val 530 535 540 Lys Gly Asp Lys
Val Gly Gln Val Asn Gly Leu Ser Val Ile Asp Leu545 550 555 560 Gly
Tyr Ala Ser Phe Gly Arg Pro Ile Arg Ile Thr Cys Cys Cys Tyr 565 570
575 Lys Gly Asn Gly Asp Ile Ile Asp Ile Gln Lys Glu Ser Asn Leu Ser
580 585 590 Gly Asn Ile His Asn Lys Ala Ile Ser Thr Leu Lys Gly Tyr
Ile Asn 595 600 605 Ser Ile Ile Gly Lys Tyr Asp Thr Leu Pro Val Asp
Phe His Leu Ser 610 615 620 Phe Glu Gln Ile Tyr Gly Thr Val Asp Gly
Asp Ser Ala Ser Val Ala625 630 635 640 Glu Ala Ile Ala Met Leu Ser
Ala Leu Ser Asn Ile Pro Val Arg Gln 645 650 655 Ser Ile Ala Val Thr
Gly Ser Ile Asn Gln Phe Gly Gln Val Gln Pro 660 665 670 Ile Gly Gly
Val Asn Glu Lys Ile Glu Gly Phe Tyr Glu Val Cys Arg 675 680 685 Tyr
Lys Lys Asp Ile Lys Asp Lys Gly Ile Leu Ile Pro Lys Ser Asn 690 695
700 Lys Glu Asn Leu Val Leu Asn Lys Glu Val Glu Glu Ala Ile Lys
Asn705 710 715 720 Gly Glu Phe Ser Ile Tyr Thr Met Glu Thr Leu Glu
Asp Ala Val Lys 725 730 735 Ile Leu Leu Gly Glu Lys Asn Leu Lys Phe
Asn Glu Leu Ile Val Glu 740 745 750 Ile Glu Lys Glu Leu Lys Lys Tyr
Asn Lys Lys Ser Lys Lys Cys Arg 755 760 765 Lys Ile Lys Leu 770
40575PRTClostridium botulinum serotype A 40Met Lys Ser Lys Lys Leu
Leu Thr Ile Leu Leu Ser Ala Ile Ile Thr1 5 10 15 Ala Ser Ser Ile
Ser Ser Val Tyr Ala Ala Glu Ser Val Gly Ile Lys 20 25 30 Ser Lys
Tyr Gln Pro Lys Thr Thr Thr Ile Phe Trp Glu Lys Gly Lys 35 40 45
Gln Asn Asn Lys Lys Ser Ala Thr Asn Ile Ala Ser Glu Gln Phe Asn 50
55 60 Asn Phe Glu Glu Ile Asn Gln Phe Phe Gln Gln Asn Ile Ser Arg
Phe65 70 75 80 Gly Leu Lys Lys Gly Ser Leu Lys Ser Thr Lys Ala Leu
Lys Asp Glu 85 90 95 Lys Gly Lys Thr His Tyr His Thr Ile Tyr Gln
Val Glu Gly Ile Pro 100 105 110 Val Tyr Tyr Gly Arg Ile Val Phe Thr
Thr Glu Lys Asp Ser Thr Ile 115 120 125 Ser Ser Ile Asn Gly Gly Val
Asp Ile Ser Phe Glu Asn Glu Asn Trp 130 135 140 Lys Asn Lys Ile Lys
Leu Ser Lys Ser Asp Ala Ile Ala Lys Ala Lys145 150 155 160 Asn Asn
Ile Lys Tyr Glu Glu Leu His Asp Ser Lys Ala Asp Leu Tyr 165 170 175
Leu Tyr Asn Phe Glu Glu Lys Pro Tyr Val Val Tyr Leu Ile Asp Leu 180
185 190 Ser Thr Asp Thr Gly Asp Trp Asn Val Phe Val Asn Ala Glu Asp
Gly 195 200 205 Ser Ile Val Asn Lys Phe Asn Asn Ile Pro Thr Leu Thr
Asn Thr Arg 210 215 220 Asp Lys Lys Phe Thr Ser Thr Lys Lys Thr Asn
Thr Lys Val Asn Lys225 230 235 240 Ser Asn Asn Leu Ile Asp Val Gln
Gly Asn Thr Ile Lys Gly Lys Gly 245 250 255 Lys Ser Ser Leu Asn Gly
Ile Val Asp Ile Asp Leu Thr Tyr Lys Asp 260 265 270 Gly Lys Tyr Tyr
Leu Lys Asn Ser Asn Lys Asn Ile Tyr Val Tyr Asp 275 280 285 Leu Asn
Asn Lys Tyr Ile Asn Thr Phe Thr Thr Pro Lys Ser Ser Ile 290 295 300
Leu Lys Ala Ser Lys Leu Val Glu Asn Asn Asn Asn Glu Phe Ile Asp305
310 315 320 Asp Lys His Ile Ile Ala Val Asp Ala Tyr Ile Asn Leu Glu
Lys Thr 325 330 335 Tyr Asp Tyr Tyr Lys Asn Lys Phe Asn Arg Asn Ser
Ile Asp Asn Lys 340 345 350 Gly Met Asn Val Glu Ala Phe Ile His His
Gly Glu Lys Tyr Ala Gly 355 360 365 Ala Glu Trp Ser Glu Asn Leu Gly
Ser Met Leu Leu Gly Asp Gly Asp 370 375 380 Gly Arg Asn Ser Ser His
Met Ser Lys Ala Leu Asp Val Val Gly His385 390 395 400 Glu Phe Ser
His Gly Val Thr Arg Lys Glu Ser Asn Leu Lys Tyr Glu 405 410 415 Asn
Glu Ser Gly Ala Leu Asn Glu Ser Phe Ser Asp Ile Met Gly Ile 420 425
430 Ala Ile Lys Gly Lys Asn Phe Lys Leu Gly Glu Asp Cys Trp Thr Pro
435 440 445 Asp Ile Glu Gly Asp Ala Ile Arg Asp Met Gln Asp Pro Ser
Lys Gly 450 455 460 Tyr Gln Pro Ala His Met Lys Asp Tyr Arg Ser Met
Asp Ile Arg Tyr465 470 475 480 Asp Asn Gly Gly Val His Val Asn Ser
Gly Ile Ile Asn His Ala Ala 485 490 495 Tyr Leu Ile Ala Asp Gly Ile
Glu Lys Leu Gly Val Glu Asn Ser Lys 500 505 510 Asp Ile Met Ala Lys
Leu Phe Tyr Thr Ala Asn Cys Tyr Glu Trp Asp 515 520 525 Glu Thr Thr
Asn Phe Ser Lys Cys Arg Asn Asp Leu Ile Lys Val Thr 530 535 540 Lys
Asp Leu Tyr Gly Glu Asn Ser Lys Tyr Val Gln Ile Val Glu Asn545 550
555 560 Ala Phe Asp Lys Val Gly Ile Thr Ala Thr Pro Gln Leu Pro Leu
565 570 575 41592PRTClostridium botulinum serotype A 41Met Lys Ser
Lys Lys Leu Leu Ala Thr Val Leu Ser Ala Val Ile Thr1 5 10 15 Phe
Ser Ala Val Ser Ala Val Ser Ala Ala Pro Val Gly Lys Glu Ser 20 25
30 Lys Ser Glu Pro Lys Ile Thr Thr Ile Ser Trp Asp Lys Ser Glu Gln
35 40 45 Asn Thr Lys Lys Asp Ala Thr Asp Ile Lys Gln Lys Lys Phe
Asn Asn 50 55 60 Ala Gln Glu Val Thr Asn Phe Phe Glu Lys Asn Ile
Ser Lys Phe Gly65 70 75 80 Val Lys Lys Gly Thr Leu Lys Ser Thr Lys
Thr Leu Lys Asp Asp Lys 85 90 95 Gly Lys Thr His Tyr His Thr Ile
Tyr Glu Val Glu Gly Ile Pro Val 100 105 110 Tyr Tyr Gly Arg Ile Val
Phe Thr Thr Glu Lys Asp Ser Thr Met Asn 115 120 125 Ser Ile Asn Gly
Arg Val Asp Thr Val Phe Glu Asn Gly Asn Trp Lys 130 135 140 Asn Lys
Ile Lys Leu Ser Lys Glu Asp Ala Ile Ala Lys Ala Lys Gly145 150 155
160 Asp Ile Lys Asp Gln Lys Ser Asn Ser Glu Lys Ala Asp Leu Tyr Leu
165 170 175 Tyr Asn Phe Glu Gly Lys Pro Tyr Val Val Tyr Leu Val Asn
Thr Met 180 185 190 Thr Asp Ser Gly Asn Trp Asn Val Phe Val Asn Ala
Glu Asp Gly Ser 195 200 205 Ile Val Asn Lys Phe Asp Thr Thr Pro Thr
Leu Val Glu Asn Lys Asp 210 215 220 Lys Lys Leu Pro Asn Ala Lys Lys
Ile Lys Asp Glu Ala Glu Lys Asn225 230 235 240 Glu Ala Lys Lys Ala
Asn Ala Ser Asn Val Asn Ser Val Thr Asp Val 245 250 255 Gln Gly Gln
Ser Val Lys Gly Met Gly Arg Thr Ser Leu Asp Gly Leu 260 265 270 Val
Asn Leu Asp Leu Thr Tyr Gly Ser Gly Arg Tyr Tyr Leu Lys Asp 275 280
285 Asn Asn Arg Lys Ile Tyr Leu Tyr Asp Leu Lys Asn Gln Val Ser Gly
290 295 300 Asp Asp Leu Tyr Arg Tyr Ile Ile Glu His Tyr Tyr Tyr Gly
Ala Pro305 310 315 320 Glu Tyr Lys Gln Arg Leu Met Ser Gln Ser Glu
Leu Val Ser Asn Ser 325 330 335 Asn Asn Asn Phe Ile Asn Asp Asn Gln
Val Asn Ser Val Asp Ala Tyr 340 345 350 Val Asn Thr Ala Lys Thr Tyr
Asp Tyr Tyr Lys Asn Lys Leu Ser Arg 355 360 365 Asn Ser Ile Asp Asn
Lys Gly Met Asn Val Asn Gly Phe Val His Val 370 375 380 Asp Lys Asn
Leu Gly Asn Ala Phe Trp Tyr Gly Pro Tyr Asp Ser Met385 390 395 400
Phe Phe Gly Asp Gly Asp Gly Val Arg Phe Ser Ala Leu Ala Lys Ser 405
410 415 Leu Asp Val Val Gly His Glu Leu Ser His Gly Val Thr Asn Lys
Gln 420 425 430 Ser Asn Leu Asn Tyr Ala Asn Glu Ser Gly Ala Leu Asn
Glu Ser Phe 435 440 445 Ser Asp Ile Met Gly Thr Ala Val Glu Gly Lys
Asn Phe Val Leu Gly 450 455 460 Glu Asp Cys Trp Ile Ala Gly Gly Val
Met Arg Asp Met Glu Asn Pro465 470 475 480 Ser Arg Gly Asn Gln Pro
Ala His Met Lys Asp Tyr Val Tyr Met Ser 485 490 495 Glu Asp Asn Gly
Gly Val His Lys Asn Ser Gly Ile Ile Asn His Ala 500 505 510 Ala Tyr
Leu Ile Ala Asp Gly Phe Glu Lys Met Gly Ala Lys Asp Ser 515 520 525
Lys Asp Ile Met Gly Lys Leu Phe Tyr Ile Ala Asn Cys Tyr Tyr Trp 530
535 540 Asp Gln Thr Thr Asp Phe Ala Lys Cys Arg Asn Asp Val Val Arg
Val545 550 555 560 Ala Lys Asp Leu Tyr Gly Glu Asn Ser Lys Glu Val
Gln Ile Val Lys 565 570 575 Asn Ala Phe Asp Lys Val Gly Val Ser Ala
Thr Pro Gln Leu Ser Leu 580 585 590 42594PRTClostridium botulinum
serotype A 42Met Lys Ser Lys Lys Leu Leu Ala Thr Val Leu Ser Ala
Val Ile Thr1 5 10 15 Phe Ser Ala Val Ser Ala Val Ser Ala Ala Pro
Val Gly Lys Glu Ser 20 25 30 Lys Ser Asp Pro Lys Thr Thr Thr Ile
Ser Trp Asp Lys Ser Glu Gln 35 40 45 Asn Ala Lys Lys Ala Thr Thr
Asp Ile Lys Gln Lys Lys Phe Asp Asn 50 55 60 Ala Gln Glu Ile Thr
Lys Phe Phe Glu Lys Asn Ile Ser Lys Phe
Gly65 70 75 80 Val Lys Lys Gly Thr Leu Asn Ser Thr Lys Thr Leu Lys
Asp Asp Lys 85 90 95 Gly Lys Thr His Tyr His Thr Ile Tyr Glu Val
Glu Gly Ile Pro Val 100 105 110 Tyr Tyr Gly Arg Ile Val Phe Thr Thr
Glu Lys Asp Ser Thr Met Asp 115 120 125 Ser Ile Asn Gly Arg Val Asp
Thr Val Phe Glu Asn Gly Asn Trp Lys 130 135 140 Asn Lys Ile Lys Leu
Ser Lys Glu Asp Ala Ile Ala Lys Ala Lys Ala145 150 155 160 Asp Ile
Lys Asn Glu Lys Ser Asn Lys Glu Lys Ala Glu Leu Tyr Leu 165 170 175
Tyr Asn Phe Glu Gly Lys Pro Tyr Val Val Tyr Leu Val Asn Ser Ile 180
185 190 Thr Asn Ser Gly Asn Trp Asp Ile Phe Val Asn Ala Glu Asp Gly
Ser 195 200 205 Ile Val Asn Lys Phe Asn Asn Thr Pro Thr Leu Leu Asp
Thr Lys Ala 210 215 220 Glu Lys Leu Pro Asn Ala Lys Lys Ile Lys Asp
Glu Ala Glu Lys Asn225 230 235 240 Glu Ala Lys Lys Ala Asn Asn Met
Asn Asn Ile Ile Asp Val Gln Gly 245 250 255 Gln Ser Val Lys Gly Leu
Gly Arg Thr Ser Leu Asp Gly Leu Val Asn 260 265 270 Ile Asn Leu Thr
Tyr Asp Asn Gly Arg Tyr Tyr Leu Lys Asp Asn Asn 275 280 285 Arg Lys
Ile Tyr Leu Tyr Asp Leu Lys Asn Gln Val Asp Leu Asp Asp 290 295 300
Leu Asn Asp Phe Tyr Asp Ser Pro Lys Gly Gly His Asn Glu Glu Leu305
310 315 320 Met Arg Arg Ser Glu Leu Val Ser Asn Ser Asn Asn Asn Phe
Val Asp 325 330 335 Asp Asn Gln Val Asn Ser Val Asp Ala Tyr Ala Asn
Met Ala Lys Ser 340 345 350 Tyr Asp Tyr Tyr Lys Asn Lys Leu Ser Arg
Asn Ser Leu Asp Asn Lys 355 360 365 Gly Met Asn Ile Lys Gly Phe Val
His Phe Asp Lys Asn Leu Gly Asn 370 375 380 Ala Phe Trp Val Gly Glu
Tyr Asp Ser Met Phe Phe Gly Asp Gly Asp385 390 395 400 Gly Val Arg
Leu Ser Pro Leu Ala Lys Ala Leu Asp Val Val Gly His 405 410 415 Glu
Leu Ser His Gly Val Thr Asn Lys Gln Ser Asp Leu Lys Tyr Glu 420 425
430 Lys Glu Ser Gly Ala Leu Asn Glu Ser Phe Ser Asp Ile Met Gly Thr
435 440 445 Ala Ile Glu Gly Lys Asn Phe Glu Ile Gly Glu Asp Cys Trp
Ile Pro 450 455 460 Ser Asp Arg Tyr Gly Glu Ile Met Arg Asp Met Lys
Asp Pro Ser Arg465 470 475 480 Gly Asn Gln Pro Ala His Met Lys Asp
Phe Arg Asp Leu Pro Val Asp 485 490 495 Glu Asp His Asp Trp Gly Gly
Val His Thr Asn Ser Gly Ile Ile Asn 500 505 510 His Ala Ala Tyr Leu
Ile Ala Asp Gly Phe Glu Lys Met Gly Glu Lys 515 520 525 Asp Ser Lys
Asp Ile Met Ala Lys Ile Phe Tyr Ile Ala Asn Cys Tyr 530 535 540 Tyr
Trp Asp Gln Ile Thr Asp Phe Ser Lys Cys Arg Asn Asp Val Val545 550
555 560 Lys Val Ala Lys Asp Leu Tyr Gly Asp Asn Ser Lys Glu Val Gln
Ile 565 570 575 Val Lys Asn Ala Phe Asp Gln Val Gly Ile Thr Ala Thr
Pro Gln Leu 580 585 590 Pro Leu43581PRTClostridium botulinum
serotype A 43Met Lys Ser Lys Lys Leu Leu Ala Thr Val Leu Ser Ala
Val Ile Thr1 5 10 15 Phe Ser Thr Val Ser Ala Val Tyr Ala Ala Pro
Val Gly Lys Glu Ser 20 25 30 Lys Val Glu Pro Lys Thr Thr Thr Ile
Thr Trp Glu Lys Asn Glu Gln 35 40 45 Asn Thr Lys Lys Ala Ala Thr
Asp Ile Thr Glu Lys Lys Phe Asn Asn 50 55 60 Ser Glu Glu Ile Thr
Lys Phe Phe Glu Lys Asn Ile Ser Lys Phe Gly65 70 75 80 Val Gln Lys
Gly Ser Leu Lys Asn Thr Lys Thr Val Lys Asp Glu Lys 85 90 95 Gly
Lys Thr Asn Tyr His Met Ile Tyr Glu Val Glu Gly Ile Pro Val 100 105
110 Tyr Tyr Gly Arg Ile Val Phe Thr Thr Glu Lys Asp Ser Ser Met Asp
115 120 125 Ser Ile Asn Gly Arg Ile Asp Thr Val Phe Glu Asn Gly Asn
Trp Lys 130 135 140 Asn Lys Ile Lys Leu Ser Lys Glu Asp Ala Ile Ala
Lys Ala Lys Asn145 150 155 160 Asp Ile Lys Asp Glu Lys Ala Thr Ser
Lys Lys Thr Asp Leu Tyr Leu 165 170 175 Tyr Asn Phe Glu Gly Lys Pro
Tyr Val Val Tyr Leu Val Asp Leu Ile 180 185 190 Thr Asp Asn Gly Ser
Trp Thr Val Phe Val Asn Ala Glu Asp Gly Ser 195 200 205 Ile Val Asn
Lys Phe Asn Asn Thr Pro Thr Leu Ile Asp Thr Lys Asp 210 215 220 Gln
Lys Leu Pro Asn Ala Lys Lys Ile Lys Asp Glu Ala Lys Lys Ala225 230
235 240 Ser Asn Ala Asn Asn Val Ile Asp Val Gln Gly Gln Ser Val Lys
Gly 245 250 255 Val Gly Lys Thr Ser Leu Asp Gly Leu Val Asn Ile Asp
Val Thr Tyr 260 265 270 Gly Asn Gly Lys Tyr Tyr Leu Lys Asp Ser Asn
Lys Asn Ile Tyr Leu 275 280 285 Tyr Asp Leu Lys Asn Gln Val Asp Glu
Tyr Asp Leu Tyr Asn Tyr Leu 290 295 300 Ser Arg Pro Asn Tyr Lys Gln
Ile Leu Met Ser Lys Ser Glu Leu Ile305 310 315 320 Ser Asn Tyr Asn
Asn Asn Phe Ile Ala Asn Asn Gln Val Asn Ser Val 325 330 335 Asp Ala
Tyr Val Asn Thr Asn Lys Thr Tyr Asp Tyr Tyr Lys Asn Lys 340 345 350
Leu Asn Arg Asn Ser Ile Asp Asn Lys Gly Met Asn Ile Asn Gly Phe 355
360 365 Val His Val Gly Arg Asn Tyr Gly Asn Ala Phe Trp Tyr Gly Pro
Tyr 370 375 380 Asp Gly Met Phe Phe Gly Asp Gly Asp Gly Ile Tyr Phe
Ser Ser Leu385 390 395 400 Ala Lys Ser Leu Asp Val Val Gly His Glu
Leu Ser His Gly Val Thr 405 410 415 Asn Lys Glu Ser Asn Leu Lys Tyr
Glu Asn Glu Ser Gly Ala Leu Asn 420 425 430 Glu Ser Phe Ser Asp Ile
Met Gly Val Ala Val Glu Gly Lys Asn Phe 435 440 445 Val Leu Gly Glu
Asp Cys Trp Val Ala Gly Gly Val Met Arg Asp Met 450 455 460 Glu Asn
Pro Ser Arg Gly Gly Gln Pro Ala His Met Lys Asp Tyr Lys465 470 475
480 Tyr Lys Thr Met Asn Asp Asp Asn Gly Gly Val His Thr Asn Ser Gly
485 490 495 Ile Ile Asn His Ala Ala Tyr Leu Val Ala Asp Gly Ile Glu
Lys Thr 500 505 510 Gly Ala Lys Asn Ser Lys Asp Ile Met Gly Lys Ile
Phe Tyr Thr Ala 515 520 525 Asn Cys Tyr Lys Trp Asp Glu Thr Thr Asn
Phe Ala Lys Cys Arg Asn 530 535 540 Asp Val Val Gln Val Thr Lys Glu
Leu Tyr Gly Glu Asn Ser Asn Tyr545 550 555 560 Val Lys Ile Val Glu
Lys Ala Phe Asp Gln Val Gly Ile Thr Ala Thr 565 570 575 Pro Gln Leu
Pro Leu 580 44580PRTClostridium botulinum serotype A 44Met Lys Ser
Lys Lys Leu Leu Ala Thr Val Leu Ser Ala Val Ile Thr1 5 10 15 Phe
Ser Thr Val Ser Ala Val Ser Ala Ala Pro Val Gly Lys Glu Ser 20 25
30 Lys Ser Glu Pro Lys Thr Thr Thr Ile Phe Trp Glu Lys Ser Glu Gln
35 40 45 Asn Thr Lys Lys Ser Thr Thr Ser Ile Thr Gln Glu Lys Phe
Ser Asn 50 55 60 Ser Asp Glu Ile Thr Lys Phe Phe Glu Lys Asn Ile
Ser Lys Phe Gly65 70 75 80 Val Lys Lys Gly Ser Leu Lys Asn Thr Lys
Ala Val Lys Asp Asp Lys 85 90 95 Gly Lys Thr His Tyr His Met Ile
Tyr Gln Val Glu Asp Ile Pro Val 100 105 110 Tyr Tyr Gly Arg Ile Val
Phe Thr Thr Lys Lys Asp Ser Ser Leu Asp 115 120 125 Ser Ile Asn Gly
Arg Ile Asp Thr Ala Phe Glu Asn Glu Ser Trp Lys 130 135 140 Asp Lys
Val Lys Leu Ser Lys Asp Asn Ala Ile Glu Lys Ala Lys Ser145 150 155
160 Ser Ile Lys Tyr Asp Asn Leu Ser Lys Ser Asp Ala Asp Leu Tyr Leu
165 170 175 Tyr Asn Phe Glu Gly Lys Pro Tyr Val Val Tyr Leu Val Asp
Leu Val 180 185 190 Thr Asp Asp Gly Asp Trp Asn Val Phe Val Asn Ala
Glu Asp Gly Ser 195 200 205 Ile Val Asn Lys Phe Asn Asn Thr Pro Thr
Leu Ile Asp Asn Lys Asp 210 215 220 Gln Lys Leu Pro Asn Ala Glu Glu
Ile Lys Arg Ser Ser Glu Lys Ala225 230 235 240 Pro Asn Ile Asn Ser
Val Ile Asn Val Asn Gly Gln Ser Thr Lys Gly 245 250 255 Gln Gly Lys
Thr Ser Leu Asp Gly Ile Val Asp Ile Asp Leu Thr Tyr 260 265 270 Lys
Asp Gly Lys Tyr Tyr Leu Lys Asp Ser Asn Lys Asn Ile Tyr Leu 275 280
285 Tyr Asn Leu Asn Asn Ser Trp Ala Pro Thr Ile Gly Ile Phe Ser Lys
290 295 300 Asp Tyr Ile Leu Arg Arg Ser Thr Leu Val Glu Asn Asn Ser
Asn Lys305 310 315 320 Phe Thr Glu Asp Lys His Val Thr Ala Val Asp
Gly Tyr Val Asn Leu 325 330 335 Ser Lys Thr Tyr Asp Tyr Tyr Lys Asn
Lys Phe Asn Arg Asn Ser Ile 340 345 350 Asp Asn Lys Gly Met Asn Val
Glu Gly Phe Ile His Thr Gly Lys Asn 355 360 365 Phe Asn Asn Ala Phe
Trp Arg Asp Asp Leu Gly Ser Met Phe Phe Gly 370 375 380 Asp Gly Asp
Gly Val Lys Phe Ser Ser Phe Ala Ser Ala Leu Asp Val385 390 395 400
Val Gly His Glu Val Ser His Gly Ile Thr Ser Lys Glu Ser Lys Leu 405
410 415 Lys Tyr Glu Lys Glu Ser Gly Ala Leu Asn Glu Ser Phe Ser Asp
Ile 420 425 430 Met Gly Val Ala Ile Glu Gly Lys Asn Phe Gln Ile Gly
Glu Asp Cys 435 440 445 Tyr Thr Pro Asn Ile Pro Gly Asp Ala Leu Arg
Asp Met Glu Asp Pro 450 455 460 Ser Lys Gly Asn Gln Pro Ala His Met
Lys Asp Phe Gln Tyr Leu Pro465 470 475 480 Asn Asp Lys Asp His Asp
Trp Gly Gly Val His Thr Asn Ser Gly Ile 485 490 495 Ile Asn His Ala
Ala Tyr Leu Ile Ala Asp Gly Met Glu Lys Ser Gly 500 505 510 Glu Lys
Asn Ser Lys Asp Ile Met Ala Lys Leu Phe Tyr Arg Ala Asn 515 520 525
Cys Tyr Lys Trp Asp Glu Thr Thr Asn Phe Ala Lys Cys Arg Asn Asp 530
535 540 Val Val Gln Val Thr Lys Asp Leu Tyr Gly Glu Asn Ser Lys Tyr
Val545 550 555 560 Lys Ile Val Glu Asn Ala Phe Asp Gln Val Gly Ile
Thr Ala Thr Pro 565 570 575 Gln Leu Pro Leu 580
451209PRTClostridium botulinum serotype A 45Met Lys Lys Lys Phe Ile
Lys Ala Leu Cys Ser Val Ala Leu Ser Cys1 5 10 15 Met Ile Cys Thr
Ser Cys Ile Thr Lys Val Ser Ala Ala Pro Thr Asn 20 25 30 Asn Thr
Lys Ile Asn Ser Asn Glu Ile Asn Ile Ser Glu Asp Ser Ser 35 40 45
Lys Ser Glu Arg Ile Pro Ser Thr Lys Ser Lys Pro Leu Gly Leu Asn 50
55 60 Ala Thr Lys Ala Asn Thr Ala Lys Tyr Ser Phe Asn Asp Leu Asn
Lys65 70 75 80 Leu Ser Asn Lys Glu Ile Leu Asp Leu Thr Ser Arg Ile
Lys Trp Asn 85 90 95 Asp Ile Ser Asp Leu Phe Gln Tyr Asn Asp Asp
Ser Tyr Ala Phe Tyr 100 105 110 Ser Asn Lys Glu Arg Val Gln Ala Leu
Ile Asp Gly Leu Tyr Glu Lys 115 120 125 Ala Ser Thr Tyr Thr Gly Thr
Asp Asp Lys Gly Ile Asp Thr Leu Val 130 135 140 Glu Ile Leu Arg Ser
Gly Phe Tyr Leu Gly Phe Tyr Asn Asp Ser Leu145 150 155 160 Lys Tyr
Leu Asn Asp Arg Ser Phe Gln Asp Lys Cys Ile Pro Ala Met 165 170 175
Leu Ala Ile Glu Asn Asn Lys Asn Phe Lys Leu Gly Glu Lys Gly Gln 180
185 190 Asp Thr Val Ile Ser Ala Leu Gly Lys Leu Ile Gly Asn Ala Ser
Cys 195 200 205 Asn Ala Glu Val Val Asn Lys Thr Val Pro Ile Leu Glu
Gln Tyr Tyr 210 215 220 Arg Glu Met Asn Lys Tyr Pro Lys Asp Lys Leu
Lys Ala Asp Ala Val225 230 235 240 Tyr Ser Leu Met Lys Glu Ile Asn
Tyr Asp Ile Ser Gln Tyr Thr Tyr 245 250 255 Asp His Asn Ile Arg Asp
Gly Lys Asn Thr Pro Trp Ser Ser Lys Ile 260 265 270 Asp Pro Phe Ile
Asn Thr Ile Ser Lys Phe Ala Gly Ile Ser Lys Val 275 280 285 Thr Glu
Asp Asn Gly Trp Ile Ile Asn Asn Gly Ile Tyr Tyr Thr Ser 290 295 300
Lys Phe Ala Ile Tyr His Ser Asn Pro Ser Ile Pro His Ser Val Ile305
310 315 320 Asp Lys Cys Leu Glu Ile Leu Pro Ala Tyr Ser Glu Gln Tyr
Tyr Ile 325 330 335 Ala Val Glu Arg Ile Lys Glu Asp Phe Asn Cys Lys
Asp Ser Lys Gly 340 345 350 Asn Val Val Asp Ile Asp Lys Leu Leu Glu
Asp Gly Lys Lys His Tyr 355 360 365 Leu Pro Lys Thr Tyr Thr Phe Asp
Asn Gly Lys Met Ile Ile Lys Ala 370 375 380 Gly Asp Lys Val Asp Glu
Ser Lys Ile Gln Arg Leu Tyr Trp Ala Ser385 390 395 400 Lys Glu Val
Lys Ser Gln Phe His Arg Ile Ile Gly Asn Asp Lys Pro 405 410 415 Leu
Glu Ala Gly Asn Ala Asp Asp Val Leu Thr Met Val Ile Tyr Asn 420 425
430 Ser Pro Glu Glu Tyr Lys Leu Asn Arg Thr Leu Tyr Gly Tyr Ser Val
435 440 445 Asp Asn Gly Gly Ile Tyr Ile Glu Gly Ile Gly Thr Phe Phe
Thr Tyr 450 455 460 Glu Arg Thr Pro Glu Glu Ser Ile Tyr Ser Leu Glu
Glu Leu Phe Arg465 470 475 480 His Glu Phe Thr His Tyr Leu Gln Gly
Arg Tyr Leu Val Pro Gly Leu 485 490 495 Phe Asn Glu Gly Asp Phe Tyr
Lys Gly Asn Ser Gly Arg Ile Thr Trp 500 505 510 Phe Glu Glu Gly Ser
Ala Glu Phe Phe Ala Gly Ser Thr Arg Thr Ser 515 520 525 Val Leu Pro
Arg Lys Ser Met Val Gly Gly Leu Ser Glu Asn Pro Lys 530 535 540 Glu
Arg Phe Ser Ala Asp Lys Ile Leu His Ser Lys Tyr Asp Asp Gly545 550
555 560 Trp Glu Phe Tyr Lys Tyr Gly Tyr Ala Phe Ser Asp Tyr Met Tyr
Asn 565 570 575 Asn Ser Lys Lys Leu Phe Ser Asp Leu Val Ser Thr Met
Lys Asn Asn 580 585 590 Asp Val Lys Gly Tyr Glu Asn Leu Ile Glu Asn
Ala Ser Lys Asp Pro 595 600 605 Asn Val Asn Lys Ser Tyr Gln Asn
His Met Gln Lys Leu Val Asp Asn 610 615 620 Tyr Asn Asn Tyr Thr Ile
Pro Leu Val Ser Asp Asp Tyr Met Lys Lys625 630 635 640 Tyr Ser Asn
Lys Ser Leu Asn Glu Ile Lys Ser Asp Ile Glu Ser Thr 645 650 655 Met
Asn Leu Thr Asn Ser Glu Ile Thr Lys Glu Ser Ser Gln Tyr Phe 660 665
670 Asp Thr Tyr Thr Leu Lys Ala Asn Tyr Thr Leu Asp Ser Asn Lys Gly
675 680 685 Glu Ile Asp Asn Trp Asn Cys Met Asn Asn Lys Val Asn Glu
Ser Leu 690 695 700 Glu Lys Leu Asn Lys Leu Gly Trp Gly Gly Tyr Lys
Thr Val Thr Ala705 710 715 720 Tyr Phe Ser Asn Pro Lys Val Asn Ser
His Asn Gln Val Glu Tyr Asn 725 730 735 Val Val Phe His Gly Leu Leu
Thr His Asn Lys Asn Phe Asn Glu Ala 740 745 750 Pro Thr Ile Lys Leu
Asp Phe Pro Lys Glu Ala Asn Thr Asp Glu Lys 755 760 765 Ile Lys Phe
Ser Ser Glu Gly Ser Thr Asp Asp Gly Lys Ile Val Ser 770 775 780 Tyr
Ala Trp Asp Leu Gly Asp Gly Glu Thr Ser Ser Glu Lys Asn Pro785 790
795 800 Thr His Val Tyr Lys Ala Pro Gly Thr Tyr Thr Val Lys Leu Thr
Val 805 810 815 Thr Asp Asp Lys Gly Leu Lys Ser Glu Lys Ser Ala Ser
Ile Asn Ile 820 825 830 Lys Lys Val Leu Thr Gly Asn Ala Val Ser Glu
Lys Glu Asn Asn Asn 835 840 845 Asp Tyr Val Asn Ala Asn Pro Val Tyr
Ser Lys Asp Leu Val Ser Gly 850 855 860 Ser Val Ser Ser Ser Asp Asp
Arg Asp Ile Phe Tyr Phe Asn Val Thr865 870 875 880 Lys Pro Ser Asp
Ile Thr Ile Asn Ala Glu Lys Ile Asn Lys Asp Lys 885 890 895 Ser Glu
Phe Thr Trp Leu Leu Phe Ser Glu Glu Asp Lys Ser Asn Tyr 900 905 910
Ile Ala Tyr Pro Asn Lys Lys Leu Glu Asn Leu Phe Tyr Ser Thr Val 915
920 925 Lys Ile Asp Lys Pro Gly Lys Tyr Tyr Leu Val Ile Tyr Lys Val
Ser 930 935 940 Gly Asp Lys Ser Asp Tyr Arg Phe Asn Ile Glu Gly Asp
Ile Ser Ala945 950 955 960 Ser Thr Lys Asp Asp Thr Asp Lys Asn Glu
Leu Val Ile Ser Glu Lys 965 970 975 Glu Asp Asn Asn Ser Phe Asp Lys
Ala Asn Arg Val Cys Lys Asn Gln 980 985 990 Ser Val Ile Ala Thr Leu
Asp Thr Asn Asp Pro Arg Asp Thr Tyr Tyr 995 1000 1005 Phe Asp Ala
Leu Thr Val Gly Asn Ile Glu Val Thr Met Glu Asn Thr 1010 1015 1020
Asp Asn Asn Ser Asn Glu Phe Asn Trp Leu Ala Tyr Ser Ser Asp Asn1025
1030 1035 1040Thr Asn Asn Tyr Ile Gly Tyr Ala Thr Lys Arg Glu Gly
Asn Lys Ile 1045 1050 1055 Ile Gly Asn Phe Lys Val Asp Lys Pro Gly
Arg Tyr Tyr Ile Val Ala 1060 1065 1070 Tyr Lys Thr Ser Ser Asn Lys
Ile Asn Tyr Lys Leu Asn Ile Lys Gly 1075 1080 1085 Asp Ile Asp Asn
Val Pro Lys Asn Asp Glu Ile Tyr Glu Lys Glu Ser 1090 1095 1100 Asn
Asp Ser Phe Glu Thr Ala Asn Lys Ile Met Leu Asn Thr Thr Val1105
1110 1115 1120Leu Gly Asn Leu Asn Gly Lys Asp Val Arg Asp Ile Tyr
Ser Phe Asp 1125 1130 1135 Ile Lys Glu Ala Lys Asp Leu Asp Ile Lys
Leu Asn Asn Leu Asn Asn 1140 1145 1150 Leu Gly Leu Ala Trp Asn Leu
Tyr Lys Glu Ser Asp Leu Asn Asn Tyr 1155 1160 1165 Ile Ala Tyr Gly
Ser Val Ser Gly Asn Thr Ile Gln Gly Lys Cys Asn 1170 1175 1180 Val
Thr Pro Gly Lys Tyr Tyr Leu Tyr Val Tyr Lys Tyr Ser Gly Asp1185
1190 1195 1200Asn Gly Asn Tyr Ser Leu Thr Ile Lys 1205
46212PRTClostridium botulinum serotype A 46Met Leu Asn Glu Phe Tyr
Thr Lys Gly Glu Glu Ile Ala Asn Ala Ile1 5 10 15 Thr His Gly Ile
Gly Ala Leu Leu Ser Ile Ala Ala Leu Val Ile Leu 20 25 30 Ile Val
Phe Ser Ala Lys Tyr Gly Asp Ala Trp Tyr Val Thr Ser Tyr 35 40 45
Ser Ile Phe Gly Ser Cys Leu Phe Ile Leu Tyr Leu Glu Ser Thr Leu 50
55 60 Tyr His Ser Leu Gln Gly Ser Lys Val Lys Lys Ile Phe Arg Ile
Phe65 70 75 80 Asp His Ser Ser Ile Phe Leu Leu Ile Ala Gly Thr Tyr
Thr Pro Phe 85 90 95 Ile Leu Thr Ser Leu Arg Asp Pro Leu Gly Trp
Thr Ile Phe Gly Ile 100 105 110 Glu Trp Gly Leu Thr Leu Ile Gly Ile
Ile Leu Lys Val Phe Thr Thr 115 120 125 Gly Lys Tyr Glu Lys Leu Ser
Thr Ala Ile Tyr Ile Phe Met Gly Trp 130 135 140 Leu Ile Met Leu His
Ala Lys Lys Leu Val Leu Val Ile Pro Arg Ile145 150 155 160 Ser Leu
Ile Tyr Leu Ile Val Gly Gly Val Ile Tyr Thr Val Gly Ala 165 170 175
Phe Leu Phe Met Leu Asp Asp Ile Pro Tyr Asn His Pro Ile Trp His 180
185 190 Leu Phe Val Ile Gly Gly Ser Val Phe His Phe Phe Ser Leu Phe
Tyr 195 200 205 Met Ile Pro Arg 210 47216PRTClostridium botulinum
serotype A 47Met Phe Lys Lys Phe Arg Asp Pro Val Ser Gly Leu Thr
His Leu Phe1 5 10 15 Gly Ala Ile Met Ser Ile Val Gly Leu Ile Val
Leu Val Asn Tyr Ser 20 25 30 Ile Phe Gln Asp Ser Pro Leu His Ile
Thr Val Phe Ala Ile Phe Gly 35 40 45 Ala Ser Leu Ile Leu Leu Tyr
Ser Ala Ser Ser Ile Tyr His Leu Val 50 55 60 Thr Ala Ser Lys Lys
Ser Ile Arg Ile Leu Arg Arg Ile Asp His Ser65 70 75 80 Met Ile Tyr
Val Leu Ile Ala Gly Ser Tyr Thr Pro Ile Cys Leu Leu 85 90 95 Ala
Leu Lys Gly Thr Phe Gly Leu Ala Met Leu Thr Ile Ile Trp Thr 100 105
110 Leu Ala Ile Ile Gly Ile Leu Val Lys Asn Phe Trp Phe Ser Ala Pro
115 120 125 Arg Trp Ile Ser Thr Gly Phe Tyr Leu Ile Met Gly Trp Leu
Ile Val 130 135 140 Val Ala Ile Phe Pro Leu Ser Lys Thr Leu Ser Ile
Gly Gly Leu Phe145 150 155 160 Trp Leu Ile Ala Gly Gly Val Ala Tyr
Ser Ile Gly Ala Val Ile Tyr 165 170 175 Gly Thr Lys Arg Pro Lys Ile
Ala Ser Lys Tyr Phe Thr Phe His Asp 180 185 190 Ile Phe His Ile Phe
Val Leu Leu Gly Ser Leu Cys His Phe Ile Leu 195 200 205 Met Leu Asn
Tyr Ile Met Tyr Met 210 215 48401PRTClostridium botulinum serotype
A 48Met Lys Lys Asn Lys Lys Trp Ile Ile Trp Thr Val Val Ile Val
Leu1 5 10 15 Val Thr Asn Ile Phe Thr Phe Leu Gly Thr Asn Leu Val
Ser Leu Tyr 20 25 30 Leu Pro Asn Gly Lys Val Ile Ile Gly Ala Asp
Gln Tyr Lys Asp Ile 35 40 45 Leu Lys Tyr Gln Lys Met Phe Leu Ile
Arg Asn Gln Ile Tyr Lys Tyr 50 55 60 Tyr Asp Gly Lys Ile Asp Glu
Ser Lys Met Val Glu Gly Ala Val Lys65 70 75 80 Gly Met Thr Glu Ser
Leu Asn Asp Pro Tyr Thr Val Phe Met Asn Ala 85 90 95 Lys Glu Tyr
Lys Glu Phe Asn Ala Gln Thr Glu Gly Asn Tyr Ser Gly 100 105 110 Val
Gly Ile Gln Ile Gln Ala Lys Asp Asp Lys Ile Ile Val Ala Ser 115 120
125 Thr Phe Glu Gly Ser Pro Ala Lys Glu Ala Gly Ile Leu Pro Lys Asp
130 135 140 Glu Ile Gln Lys Val Asn Asn Thr Thr Val Ser Gly Lys Glu
Leu Glu145 150 155 160 Lys Ala Val Ser Ile Met Lys Gly Lys Glu Gly
Thr Asp Val Lys Leu 165 170 175 Gln Leu Tyr Arg Lys Glu Lys Gly Ser
Phe Glu Val Thr Leu Lys Arg 180 185 190 Lys Lys Ile Asp Ile Pro Thr
Ile Lys Ser Glu Met Ile Asp Asn Asn 195 200 205 Ile Gly Tyr Ile Gln
Val Ser Met Phe Asp Glu Asn Thr Ser Lys Asn 210 215 220 Phe Lys Asn
Ala Leu Asn Asp Leu Lys Asp Lys Gly Met Lys Ser Leu225 230 235 240
Leu Leu Asp Leu Arg Gly Asn Pro Gly Gly Leu Leu Asp Glu Cys Ile 245
250 255 Asn Met Ala Ser Asn Phe Ile Glu Lys Gly Lys Val Val Val Ser
Thr 260 265 270 Ile Asp Lys Tyr Gly Ser Lys Lys Glu Tyr Lys Ser Lys
Gly Gly Asp 275 280 285 Phe Ile Gly Phe Pro Val Thr Ile Leu Val Asp
Glu Gly Ser Ala Ser 290 295 300 Ala Ser Glu Val Phe Leu Gly Ala Met
Lys Asp Tyr Asn Val Ala Thr305 310 315 320 Ser Ile Gly Lys Lys Thr
Phe Gly Lys Gly Val Val Gln Thr Ile Ile 325 330 335 Glu Thr Gly Asp
Asn Thr Ala Leu Lys Val Thr Ile Ser Lys Tyr Tyr 340 345 350 Ser Pro
Lys Gly Val Asn Ile Asn His Lys Gly Ile Thr Pro Asp Met 355 360 365
Glu Ile Asp Tyr Pro Glu Glu Leu Arg Lys Lys Glu Tyr Asp Arg Lys 370
375 380 Val Asp Pro Gln Phe Asn Lys Ala Leu Asn Ile Ala Lys Ser Lys
Ile385 390 395 400 Arg49311PRTClostridium botulinum serotype A
49Met Ser Lys Asp Ile Gly Lys Arg Leu Ile Ile Leu Leu Ser Ile Gly1
5 10 15 Val Thr Ile Leu Val Ile Thr Tyr Ala Tyr Ile Tyr Thr Lys Pro
Asn 20 25 30 Ala Tyr Glu Val Leu Val Asn Asn Asn Pro Val Ala Tyr
Val Lys Asn 35 40 45 Lys Glu Asp Phe Asn Lys Ile Tyr Lys Glu Val
Glu Asn Asn Ile Lys 50 55 60 Lys Arg Phe Asn Leu Asp Met Arg Asn
Asn Ile Asn Phe Glu Asn Ile65 70 75 80 Lys Val Lys Gly Asp Ile Leu
Thr Ser Asn Asp Phe Ile Lys Lys Ser 85 90 95 Ile Leu Glu Asn Ser
Asn Ile Lys Val Thr Ala Phe Lys Val Lys Phe 100 105 110 Gln Asp Glu
Phe Ile Gly Ile Leu Ser Asn Lys Lys Glu Ile Gln Asp 115 120 125 Leu
Ser Lys Ile Ile Ser Lys Lys Tyr Ser Val Asn Ile Ile Asn His 130 135
140 Ile Lys Ile Lys Glu Glu Thr Ile Ser Val Glu Glu Ile Asn Thr
Ile145 150 155 160 Asp Glu Leu Ala Ile Asn Ile Ser Lys Ser Gln Lys
Leu Gln Asn Phe 165 170 175 Met Asn Ser Lys Arg Leu Ser Arg Gly Asp
Ile Asn Glu Glu Ile Ala 180 185 190 Leu Ala Met Pro Thr Asn Gly Cys
Ile Thr Ser Lys Phe Gly Lys Arg 195 200 205 Trp Gly Lys Phe His Lys
Gly Leu Asp Ile Gly Ala Pro Asn Gly Thr 210 215 220 Asp Ile Tyr Ser
Ser Leu Asp Gly Lys Val Ile Tyr Ser Gly Trp Glu225 230 235 240 Glu
Gly Tyr Gly Lys Val Ile Lys Ile Gln His Ser Ser Glu Leu Ile 245 250
255 Thr Ile Tyr Ala His Cys Ser Asn Leu Tyr Val Lys Val Gly Gln Tyr
260 265 270 Val Lys Lys Gly Glu Lys Ile Gly Glu Val Gly Ser Thr Gly
Arg Ser 275 280 285 Thr Gly Pro His Val His Phe Glu Leu Arg Lys Asn
Asn Glu Pro Cys 290 295 300 Asn Pro Leu Thr Tyr Ile Lys305 310
501262PRTArtificial SequenceBoNT/E-DiA 50Met Pro Lys Ile Asn Ser
Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg1 5 10 15 Thr Ile Leu Tyr
Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30 Phe Asn
Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45
Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50
55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu
Lys65 70 75 80 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg
Ile Asn Asn 85 90 95 Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu
Ser Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp
Asn Gln Phe His Ile Gly Asp 115 120 125 Ala Ser Ala Val Glu Ile Lys
Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135 140 Leu Pro Asn Val Ile
Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr145 150 155 160 Asn Ser
Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175
Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180
185 190 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala
Leu 195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu
Tyr Gly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln
Lys Gln Asn Pro Leu225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn
Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile
Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu
Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln
Val Ser Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300
Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305
310 315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe
Thr Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg
Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn
Leu Leu Asn Asp Ser Ile 355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn
Ile Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn
Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr385 390 395 400 Gly Arg Gly
Leu Val Lys Lys Ile Ile Arg Phe Cys Val Arg Gly Ile 405 410 415 Ile
Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys Ala Leu 420 425
430 Asn Asp Leu Cys Ile Glu Ile Asn Asn Gly Glu Leu Phe Phe Val Ala
435 440 445 Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile Asn Thr Pro Lys
Glu Ile 450 455 460 Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr Glu Asn
Asp Leu Asp Gln465 470 475 480 Val Ile Leu Asn Phe Asn Ser Glu Ser
Ala Pro Gly Leu Ser Asp Glu 485 490 495 Lys Leu Asn Leu Thr Ile Gln
Asn Asp Ala Tyr Ile Pro Lys Tyr Asp 500 505 510 Ser Asn Gly Thr Ser
Asp Ile Glu Gln His Asp Val Asn Glu Leu Asn 515 520 525 Val Phe Phe
Tyr Leu Asp Ala Gln Lys Val Pro Glu Gly Glu Asn Asn 530 535 540 Val
Asn Leu Thr Ser Ser
Ile Asp Thr Ala Leu Leu Glu Gln Pro Lys545 550 555 560 Ile Tyr Thr
Phe Phe Ser Ser Glu Phe Ile Asn Asn Val Asn Lys Pro 565 570 575 Val
Gln Ala Ala Leu Phe Val Ser Trp Ile Gln Gln Val Leu Val Asp 580 585
590 Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr Val Asp Lys Ile Ala Asp
595 600 605 Ile Ser Ile Val Val Pro Tyr Ile Gly Leu Ala Leu Asn Ile
Gly Asn 610 615 620 Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala Leu Glu
Leu Leu Gly Ala625 630 635 640 Gly Ile Leu Leu Glu Phe Glu Pro Glu
Leu Leu Ile Pro Thr Ile Leu 645 650 655 Val Phe Thr Ile Lys Ser Phe
Leu Gly Ser Ser Asp Asn Lys Asn Lys 660 665 670 Val Ile Lys Ala Ile
Asn Asn Ala Leu Lys Glu Arg Asp Glu Lys Trp 675 680 685 Lys Glu Val
Tyr Ser Phe Ile Val Ser Asn Trp Met Thr Lys Ile Asn 690 695 700 Thr
Gln Phe Asn Lys Arg Lys Glu Gln Met Tyr Gln Ala Leu Gln Asn705 710
715 720 Gln Val Asn Ala Ile Lys Thr Ile Ile Glu Ser Lys Tyr Asn Ser
Tyr 725 730 735 Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn Lys Tyr Asp
Ile Lys Gln 740 745 750 Ile Glu Asn Glu Leu Asn Gln Lys Val Ser Ile
Ala Met Asn Asn Ile 755 760 765 Asp Arg Phe Leu Thr Glu Ser Ser Ile
Ser Tyr Leu Met Lys Leu Ile 770 775 780 Asn Glu Val Lys Ile Asn Lys
Leu Arg Glu Tyr Asp Glu Asn Val Lys785 790 795 800 Thr Tyr Leu Leu
Asn Tyr Ile Ile Gln His Gly Ser Ile Leu Gly Glu 805 810 815 Ser Gln
Gln Glu Leu Asn Ser Met Val Thr Asp Thr Leu Asn Asn Ser 820 825 830
Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp Asp Lys Ile Leu Ile Ser 835
840 845 Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser Ser Ser Val Leu
Asn 850 855 860 Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp Thr Ser Gly
Tyr Asp Ser865 870 875 880 Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys
Tyr Pro Thr Asn Lys Asn 885 890 895 Gln Phe Gly Ile Tyr Asn Asp Lys
Leu Ser Glu Val Asn Ile Ser Gln 900 905 910 Asn Asp Tyr Ile Ile Tyr
Asp Asn Lys Tyr Lys Asn Phe Ser Ile Ser 915 920 925 Phe Trp Val Arg
Ile Pro Asn Tyr Asp Asn Lys Ile Val Asn Val Asn 930 935 940 Asn Glu
Tyr Thr Ile Ile Asn Cys Met Arg Asp Asn Asn Ser Gly Trp945 950 955
960 Lys Val Ser Leu Asn His Asn Glu Ile Ile Trp Thr Leu Gln Asp Asn
965 970 975 Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn Tyr Gly Asn Ala
Asn Gly 980 985 990 Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr
Ile Thr Asn Asp 995 1000 1005 Arg Leu Gly Asp Ser Lys Leu Tyr Ile
Asn Gly Asn Leu Ile Asp Gln 1010 1015 1020 Lys Ser Ile Leu Asn Leu
Gly Asn Ile His Val Ser Asp Asn Ile Leu1025 1030 1035 1040Phe Lys
Ile Val Asn Cys Ser Tyr Thr Arg Tyr Ile Gly Ile Arg Tyr 1045 1050
1055 Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu Thr Glu Ile Gln Thr
Leu 1060 1065 1070 Tyr Ser Asn Glu Pro Asn Thr Asn Ile Leu Lys Asp
Phe Trp Gly Asn 1075 1080 1085 Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr
Leu Leu Asn Val Leu Lys Pro 1090 1095 1100 Asn Asn Phe Ile Asp Arg
Arg Lys Asp Ser Thr Leu Ser Ile Asn Asn1105 1110 1115 1120Ile Arg
Ser Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser Gly Ile Lys 1125 1130
1135 Val Lys Ile Gln Arg Val Asn Asn Ser Ser Thr Asn Asp Asn Leu
Val 1140 1145 1150 Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala
Ser Lys Thr His 1155 1160 1165 Leu Phe Pro Leu Tyr Ala Asp Thr Ala
Thr Thr Asn Lys Glu Lys Thr 1170 1175 1180 Ile Lys Ile Ser Ser Ser
Gly Asn Arg Phe Asn Gln Val Val Val Met1185 1190 1195 1200Asn Ser
Val Gly Asn Asn Cys Thr Met Asn Phe Lys Asn Asn Asn Gly 1205 1210
1215 Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr Val Val Ala
Ser 1220 1225 1230 Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr Asn
Ser Asn Gly Cys 1235 1240 1245 Phe Trp Asn Phe Ile Ser Glu Glu His
Gly Trp Gln Glu Lys 1250 1255 1260 511306PRTArtificial
SequenceBoNT/B-DiA 51Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn
Asp Pro Ile Asp Asn1 5 10 15 Asn Asn Ile Ile Met Met Glu Pro Pro
Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile
Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr Thr Phe Gly
Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn
Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn65 70 75 80 Thr
Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90
95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile
100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu
Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys
Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile
Phe Ala Asn Leu Ile Ile145 150 155 160 Phe Gly Pro Gly Pro Val Leu
Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe
Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe
Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn
Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215
220 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu
Tyr225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn
Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala
Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile
Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln
Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val
Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr305 310 315 320 Lys
Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330
335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu
340 345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys
Ile Lys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro
Val Lys Ile Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile
Glu Glu Gly Phe Asn Ile385 390 395 400 Ser Asp Lys Asp Met Glu Lys
Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr
Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln
Met Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser 435 440 445 Leu
Asp Lys Gly Tyr Asn Lys Ala Leu Asn Asp Leu Cys Ile Asp Val 450 455
460 Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser
Asp465 470 475 480 Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr
Gln Ser Asn Tyr 485 490 495 Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu
Ile Leu Asp Thr Asp Leu 500 505 510 Ile Ser Lys Ile Glu Leu Pro Ser
Glu Asn Thr Glu Ser Leu Thr Asp 515 520 525 Phe Asn Val Asp Val Pro
Val Tyr Glu Lys Gln Pro Ala Ile Lys Lys 530 535 540 Ile Phe Thr Asp
Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln Thr545 550 555 560 Phe
Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp Asp 565 570
575 Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp Tyr
580 585 590 Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala
Gly Trp 595 600 605 Val Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala
Asn Lys Ser Asn 610 615 620 Thr Met Asp Lys Ile Ala Asp Ile Ser Leu
Ile Val Pro Tyr Ile Gly625 630 635 640 Leu Ala Leu Asn Val Gly Asn
Glu Thr Ala Lys Gly Asn Phe Glu Asn 645 650 655 Ala Phe Glu Ile Ala
Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro Glu 660 665 670 Leu Leu Ile
Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile Asp 675 680 685 Asn
Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys Arg 690 695
700 Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp
Leu705 710 715 720 Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu
Gly Met Tyr Lys 725 730 735 Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu
Glu Ile Ile Lys Tyr Arg 740 745 750 Tyr Asn Ile Tyr Ser Glu Lys Glu
Lys Ser Asn Ile Asn Ile Asp Phe 755 760 765 Asn Asp Ile Asn Ser Lys
Leu Asn Glu Gly Ile Asn Gln Ala Ile Asp 770 775 780 Asn Ile Asn Asn
Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met Lys785 790 795 800 Lys
Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn Thr 805 810
815 Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr Leu
820 825 830 Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr
Leu Lys 835 840 845 Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn
Asp Thr Ile Leu 850 855 860 Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu
Ile Leu Asn Asn Ile Ile865 870 875 880 Leu Asn Leu Arg Tyr Lys Asp
Asn Asn Leu Ile Asp Leu Ser Gly Tyr 885 890 895 Gly Ala Lys Val Glu
Val Tyr Asp Gly Val Glu Leu Asn Asp Lys Asn 900 905 910 Gln Phe Lys
Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr Gln 915 920 925 Asn
Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val Ser 930 935
940 Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn
Tyr945 950 955 960 Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys
Asn Asn Ser Gly 965 970 975 Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile
Ile Trp Thr Leu Ile Asp 980 985 990 Ile Asn Gly Lys Thr Lys Ser Val
Phe Phe Glu Tyr Asn Ile Arg Glu 995 1000 1005 Asp Ile Ser Glu Tyr
Ile Asn Arg Trp Phe Phe Val Thr Ile Thr Asn 1010 1015 1020 Asn Leu
Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu Ser Asn1025 1030
1035 1040Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly Glu
Ile Ile 1045 1050 1055 Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln
Phe Ile Trp Met Lys 1060 1065 1070 Tyr Phe Ser Ile Phe Asn Thr Glu
Leu Ser Gln Ser Asn Ile Glu Glu 1075 1080 1085 Arg Tyr Lys Ile Gln
Ser Tyr Ser Glu Tyr Leu Lys Asp Phe Trp Gly 1090 1095 1100 Asn Pro
Leu Met Tyr Asn Lys Glu Tyr Tyr Met Phe Asn Ala Gly Asn1105 1110
1115 1120Lys Asn Ser Tyr Ile Lys Leu Lys Lys Asp Ser Pro Val Gly
Glu Ile 1125 1130 1135 Leu Thr Arg Ser Lys Tyr Asn Gln Asn Ser Lys
Tyr Ile Asn Tyr Arg 1140 1145 1150 Asp Leu Tyr Ile Gly Glu Lys Phe
Ile Ile Arg Arg Lys Ser Asn Ser 1155 1160 1165 Gln Ser Ile Asn Asp
Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu 1170 1175 1180 Asp Phe
Phe Asn Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys Tyr1185 1190
1195 1200Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser
Asp Ser 1205 1210 1215 Asp Glu Phe Tyr Asn Thr Ile Gln Ile Lys Glu
Tyr Asp Glu Gln Pro 1220 1225 1230 Thr Tyr Ser Cys Gln Leu Leu Phe
Lys Lys Asp Glu Glu Ser Thr Asp 1235 1240 1245 Glu Ile Gly Leu Ile
Gly Ile His Arg Phe Tyr Glu Ser Gly Ile Val 1250 1255 1260 Phe Glu
Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr Leu Lys1265 1270
1275 1280Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys Asn
Trp Gln 1285 1290 1295 Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1300
1305 521299PRTArtificial SequenceBoNT/C1-DiA 52Met Pro Ile Thr Ile
Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn1 5 10 15 Lys Asn Ile
Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30 Pro
Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40
45 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val
50 55 60 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser
Thr Asp65 70 75 80 Ser Asp Lys Asp Pro Phe Leu Lys Glu Ile Ile Lys
Leu Phe Lys Arg 85 90 95 Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu
Ile Tyr Arg Leu Ser Thr 100 105 110 Asp Ile Pro Phe Pro Gly Asn Asn
Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125 Phe Asp Val Asp Phe Asn
Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 Asn Trp Val Lys
Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly145 150 155 160 Pro
Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170
175 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile
180 185 190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr
Asn Asp 195 200 205 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys
Met Asp Pro Ile 210 215 220 Leu Ile Leu Met His Glu Leu Asn His Ala
Met His Asn Leu Tyr Gly225 230 235 240 Ile Ala Ile Pro Asn Asp Gln
Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255 Phe Tyr Ser Gln Tyr
Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270 Phe Gly Gly
Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275
280 285 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg
Leu 290 295 300 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys
Tyr Ile Gly305 310 315 320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr
Arg Phe Val Val Glu Ser 325 330 335 Ser Gly Glu Val Thr Val Asn Arg
Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350 Glu Leu Thr Gln Ile Phe
Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 Val Gln Asn Arg
Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 Ala Asn
Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn385 390 395
400 Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser
405 410 415 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu
Tyr Leu 420 425 430 Phe Thr Lys Phe Cys Val Arg Gly Ile Ile Thr Ser
Lys Thr Lys Ser 435 440 445 Leu Asp Lys Gly Tyr Asn Lys Ala Leu Asn
Asp Leu Cys Arg Glu Leu 450 455 460 Leu Val Lys Asn Thr Asp Leu Pro
Phe Ile Gly Asp Ile Ser Asp Val465 470 475 480 Lys Thr Asp Ile Phe
Leu Arg Lys Asp Ile Asn Glu Glu Thr Glu Val 485 490 495 Ile Tyr Tyr
Pro Asp Asn Val Ser Val Asp Gln Val Ile Leu Ser Lys 500 505 510 Asn
Thr Ser Glu His Gly Gln Leu Asp Leu Leu Tyr Pro Ser Ile Asp 515 520
525 Ser Glu Ser Glu Ile Leu Pro Gly Glu Asn Gln Val Phe Tyr Asp Asn
530 535 540 Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu
Glu Ser545 550 555 560 Gln Lys Leu Ser Asp Asn Val Glu Asp Phe Thr
Phe Thr Arg Ser Ile 565 570 575 Glu Glu Ala Leu Asp Asn Ser Ala Lys
Val Tyr Thr Tyr Phe Pro Thr 580 585 590 Leu Ala Asn Lys Val Asn Ala
Gly Val Gln Gly Gly Leu Phe Leu Met 595 600 605 Trp Ala Asn Asp Val
Val Glu Asp Phe Thr Thr Asn Ile Leu Arg Lys 610 615 620 Asp Thr Leu
Asp Lys Ile Ser Asp Val Ser Ala Ile Ile Pro Tyr Ile625 630 635 640
Gly Pro Ala Leu Asn Ile Ser Asn Ser Val Arg Arg Gly Asn Phe Thr 645
650 655 Glu Ala Phe Ala Val Thr Gly Val Thr Ile Leu Leu Glu Ala Phe
Pro 660 665 670 Glu Phe Thr Ile Pro Ala Leu Gly Ala Phe Val Ile Tyr
Ser Lys Val 675 680 685 Gln Glu Arg Asn Glu Ile Ile Lys Thr Ile Asp
Asn Cys Leu Glu Gln 690 695 700 Arg Ile Lys Arg Trp Lys Asp Ser Tyr
Glu Trp Met Met Gly Thr Trp705 710 715 720 Leu Ser Arg Ile Ile Thr
Gln Phe Asn Asn Ile Ser Tyr Gln Met Tyr 725 730 735 Asp Ser Leu Asn
Tyr Gln Ala Gly Ala Ile Lys Ala Lys Ile Asp Leu 740 745 750 Glu Tyr
Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln 755 760 765
Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met 770
775 780 Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu
Phe785 790 795 800 Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn
Glu Phe Asp Arg 805 810 815 Asn Thr Lys Ala Lys Leu Ile Asn Leu Ile
Asp Ser His Asn Ile Ile 820 825 830 Leu Val Gly Glu Val Asp Lys Leu
Lys Ala Lys Val Asn Asn Ser Phe 835 840 845 Gln Asn Thr Ile Pro Phe
Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu 850 855 860 Leu Lys Asp Ile
Ile Asn Glu Tyr Phe Asn Asn Ile Asn Asp Ser Lys865 870 875 880 Ile
Leu Ser Leu Gln Asn Arg Lys Asn Thr Leu Val Asp Thr Ser Gly 885 890
895 Tyr Asn Ala Glu Val Ser Glu Glu Gly Asp Val Gln Leu Asn Pro Ile
900 905 910 Phe Pro Phe Asp Phe Lys Leu Gly Ser Ser Gly Glu Asp Arg
Gly Lys 915 920 925 Val Ile Val Thr Gln Asn Glu Asn Ile Val Tyr Asn
Ser Met Tyr Glu 930 935 940 Ser Phe Ser Ile Ser Phe Trp Ile Arg Ile
Asn Lys Trp Val Ser Asn945 950 955 960 Leu Pro Gly Tyr Thr Ile Ile
Asp Ser Val Lys Asn Asn Ser Gly Trp 965 970 975 Ser Ile Gly Ile Ile
Ser Asn Phe Leu Val Phe Thr Leu Lys Gln Asn 980 985 990 Glu Asp Ser
Glu Gln Ser Ile Asn Phe Ser Tyr Asp Ile Ser Asn Asn 995 1000 1005
Ala Pro Gly Tyr Asn Lys Trp Phe Phe Val Thr Val Thr Asn Asn Met
1010 1015 1020 Met Gly Asn Met Lys Ile Tyr Ile Asn Gly Lys Leu Ile
Asp Thr Ile1025 1030 1035 1040Lys Val Lys Glu Leu Thr Gly Ile Asn
Phe Ser Lys Thr Ile Thr Phe 1045 1050 1055 Glu Ile Asn Lys Ile Pro
Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp 1060 1065 1070 Asn Ile Asn
Met Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu 1075 1080 1085
Asp Gly Lys Asp Ile Asn Ile Leu Phe Asn Ser Leu Gln Tyr Thr Asn
1090 1095 1100 Val Val Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr Asn
Lys Glu Tyr1105 1110 1115 1120Tyr Met Val Asn Ile Asp Tyr Leu Asn
Arg Tyr Met Tyr Ala Asn Ser 1125 1130 1135 Arg Gln Ile Val Phe Asn
Thr Arg Arg Asn Asn Asn Asp Phe Asn Glu 1140 1145 1150 Gly Tyr Lys
Ile Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn Asp Thr 1155 1160 1165
Arg Val Arg Gly Gly Asp Ile Leu Tyr Phe Asp Met Thr Ile Asn Asn
1170 1175 1180 Lys Ala Tyr Asn Leu Phe Met Lys Asn Glu Thr Met Tyr
Ala Asp Asn1185 1190 1195 1200His Ser Thr Glu Asp Ile Tyr Ala Ile
Gly Leu Arg Glu Gln Thr Lys 1205 1210 1215 Asp Ile Asn Asp Asn Ile
Ile Phe Gln Ile Gln Pro Met Asn Asn Thr 1220 1225 1230 Tyr Tyr Tyr
Ala Ser Gln Ile Phe Lys Ser Asn Phe Asn Gly Glu Asn 1235 1240 1245
Ile Ser Gly Ile Cys Ser Ile Gly Thr Tyr Arg Phe Arg Leu Gly Gly
1250 1255 1260 Asp Trp Tyr Arg His Asn Tyr Leu Val Pro Thr Val Lys
Gln Gly Asn1265 1270 1275 1280Tyr Ala Ser Leu Leu Glu Ser Thr Ser
Thr His Trp Gly Phe Val Pro 1285 1290 1295 Val Ser
Glu531287PRTArtificial SequenceBoNT/D-DiA 53Met Thr Trp Pro Val Lys
Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp1 5 10 15 Asn Asp Ile Leu
Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30 Pro Val
Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45
Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50
55 60 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr
Asp65 70 75 80 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu
Phe Lys Arg 85 90 95 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile
Asn Tyr Leu Val Val 100 105 110 Gly Ser Pro Phe Met Gly Asp Ser Ser
Thr Pro Glu Asp Thr Phe Asp 115 120 125 Phe Thr Arg His Thr Thr Asn
Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140 Ser Trp Lys Val Thr
Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly145 150 155 160 Pro Leu
Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175
Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180
185 190 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser
Asn 195 200 205 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met
Asp Pro Val 210 215 220 Ile Ala Leu Met His Glu Leu Thr His Ser Leu
His Gln Leu Tyr Gly225 230 235 240 Ile Asn Ile Pro Ser Asp Lys Arg
Ile Arg Pro Gln Val Ser Glu Gly 245 250 255 Phe Phe Ser Gln Asp Gly
Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270 Phe Gly Gly Leu
Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275 280 285 Leu Arg
Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300
Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp305
310 315 320 Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys
Asp Asn 325 330 335 Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn
Ser Leu Tyr Ser 340 345 350 Asp Leu Thr Asn Val Met Ser Glu Val Val
Tyr Ser Ser Gln Tyr Asn 355 360 365 Val Lys Asn Arg Thr His Tyr Phe
Ser Arg His Tyr Leu Pro Val Phe 370 375 380 Ala Asn Ile Leu Asp Asp
Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn385 390 395 400 Leu Thr Asn
Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415 Arg
Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425
430 Phe Thr Lys Val Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser
435 440 445 Leu Asp Lys Gly Tyr Asn Lys Ala Leu Asn Asp Leu Cys Ile
Lys Val 450 455 460 Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys Asp
Ser Ile Ser Gln465 470 475 480 Glu Ile Phe Glu Asn Lys Ile Ile Thr
Asp Glu Thr Asn Val Gln Asn 485 490 495 Tyr Ser Asp Lys Phe Ser Leu
Asp Glu Ser Ile Leu Asp Gly Gln Val 500 505 510 Pro Ile Asn Pro Glu
Ile Val Asp Pro Leu Leu Pro Asn Val Asn Met 515 520 525 Glu Pro Leu
Asn Leu Pro Gly Glu Glu Ile Val Phe Tyr Asp Asp Ile 530 535 540 Thr
Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln545 550
555 560 Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu Thr Thr Ser Val
Glu 565 570 575 Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr Phe Leu
Pro Ser Leu 580 585 590 Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly
Leu Phe Leu Asn Trp 595 600 605 Ala Asn Glu Val Val Glu Asp Phe Thr
Thr Asn Ile Met Lys Lys Asp 610 615 620 Thr Leu Asp Lys Ile Ser Asp
Val Ser Val Ile Ile Pro Tyr Ile Gly625 630 635 640 Pro Ala Leu Asn
Ile Gly Asn Ser Ala Leu Arg Gly Asn Phe Asn Gln 645 650 655 Ala Phe
Ala Thr Ala Gly Val Ala Phe Leu Leu Glu Gly Phe Pro Glu 660 665 670
Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe Tyr Ser Ser Ile Gln 675
680 685 Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn Cys Leu Glu Gln
Arg 690 695 700 Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met Val Ser
Asn Trp Leu705 710 715 720 Ser Arg Ile Thr Thr Gln Phe Asn His Ile
Asn Tyr Gln Met Tyr Asp 725 730 735 Ser Leu Ser Tyr Gln Ala Asp Ala
Ile Lys Ala Lys Ile Asp Leu Glu 740 745 750 Tyr Lys Lys Tyr Ser Gly
Ser Asp Lys Glu Asn Ile Lys Ser Gln Val 755 760 765 Glu Asn Leu Lys
Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn 770 775 780 Asn Ile
Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys785 790 795
800 Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn Lys Phe Asp Leu Arg
805 810 815 Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser His Asn Ile
Ile Leu 820 825 830 Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val Asn
Glu Ser Phe Glu 835 840 845 Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr
Thr Asn Asn Ser Leu Leu 850 855 860 Lys Asp Ile Ile Asn Glu Tyr Phe
Asn Ser Ile Asn Asp Ser Lys Ile865 870 875 880 Leu Ser Leu Gln Asn
Lys Lys Asn Ala Leu Val Asp Thr Ser Gly Tyr 885 890 895 Asn Ala Glu
Val Arg Val Gly Asp Asn Val Gln Leu Asn Thr Ile Tyr 900 905 910 Thr
Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp Lys Ile Ile Val Asn 915 920
925 Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr Glu Asn Ser Ser Val
930 935 940 Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr Asn Ser His
Asn Glu945 950 955 960 Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser
Gly Trp Lys Leu Cys 965 970 975 Ile Arg Asn Gly Asn Ile Glu Trp Ile
Leu Gln Asp Val Asn Arg Lys 980 985 990 Tyr Lys Ser Leu Ile Phe Asp
Tyr Ser Glu Ser Leu Ser His Thr Gly 995 1000 1005 Tyr Thr Asn Lys
Trp Phe Phe Val Thr Ile Thr Asn Asn Ile Met Gly 1010 1015 1020 Tyr
Met Lys Leu Tyr Ile Asn Gly Glu Leu Lys Gln Ser Gln Lys Ile1025
1030 1035 1040Glu Asp Leu Asp Glu Val Lys Leu Asp Lys Thr Ile Val
Phe Gly Ile 1045 1050 1055 Asp Glu Asn Ile Asp Glu Asn Gln Met Leu
Trp Ile Arg Asp Phe Asn 1060 1065 1070 Ile Phe Ser Lys Glu Leu Ser
Asn Glu Asp Ile Asn Ile Val Tyr Glu 1075 1080 1085 Gly Gln Ile Leu
Arg Asn Val Ile Lys Asp Tyr Trp Gly Asn Pro Leu 1090 1095 1100 Lys
Phe Asp Thr Glu Tyr Tyr Ile Ile Asn Asp Asn Tyr Ile Asp Arg1105
1110 1115 1120Tyr Ile Ala Pro Glu Ser Asn Val Leu Val Leu Val Gln
Tyr Pro Asp 1125 1130 1135 Arg Ser Lys Leu Tyr Thr Gly Asn Pro Ile
Thr Ile Lys Ser Val Ser 1140 1145 1150 Asp Lys Asn Pro Tyr Ser Arg
Ile Leu Asn Gly Asp Asn Ile Ile Leu 1155 1160 1165 His Met Leu Tyr
Asn Ser Arg Lys Tyr Met Ile Ile Arg Asp Thr Asp 1170 1175 1180 Thr
Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser Gln Asn Cys Val Tyr1185
1190 1195 1200Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr Gly Ile
Gly Ile Phe 1205 1210 1215 Ser Ile Lys Asn Ile Val Ser Lys Asn Lys
Tyr Cys Ser Gln Ile Phe 1220 1225 1230 Ser Ser Phe Arg Glu Asn Thr
Met Leu Leu Ala Asp Ile Tyr Lys Pro 1235 1240 1245 Trp Arg Phe Ser
Phe Lys Asn Ala Tyr Thr Pro Val Ala Val Thr Asn 1250 1255 1260 Tyr
Glu Thr Lys Leu Leu Ser Thr Ser Ser Phe Trp Lys Phe Ile Ser1265
1270 1275 1280Arg Asp Pro Gly Trp Val Glu 1285 541282PRTArtificial
SequenceBoNT/F-DiA 54Met Pro Val Ala Ile
Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp1 5 10 15 Asp Thr Ile
Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr
Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40
45 Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser
50 55 60 Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu
Thr Thr65 70 75 80 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile
Lys Leu Phe Lys 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val
Leu Leu Gln Glu Ile Ser 100 105 110 Tyr Ala Lys Pro Tyr Leu Gly Asn
Asp His Thr Pro Ile Asp Glu Phe 115 120 125 Ser Pro Val Thr Arg Thr
Thr Ser Val Asn Ile Lys Leu Ser Thr Asn 130 135 140 Val Glu Ser Ser
Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro145 150 155 160 Asp
Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro 165 170
175 Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile
180 185 190 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile
Ser Gly 195 200 205 Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp
Pro Ala Ile Ser 210 215 220 Leu Ala His Glu Leu Ile His Ala Leu His
Gly Leu Tyr Gly Ala Arg225 230 235 240 Gly Val Thr Tyr Glu Glu Thr
Ile Glu Val Lys Gln Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro
Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270 Gln Asp Leu
Asn Ile Ile Thr Ser Ala Met Lys Glu Lys Ile Tyr Asn 275 280 285 Asn
Leu Leu Ala Asn Tyr Glu Lys Ile Ala Thr Arg Leu Ser Glu Val 290 295
300 Asn Ser Ala Pro Pro Glu Tyr Asp Ile Asn Glu Tyr Lys Asp Tyr
Phe305 310 315 320 Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly
Ser Tyr Thr Val 325 330 335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys
Lys Leu Tyr Ser Phe Thr 340 345 350 Glu Ser Asp Leu Ala Asn Lys Phe
Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365 Phe Ile Lys Tyr Glu Phe
Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380 Ile Tyr Thr Val
Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn385 390 395 400 Asn
Arg Gly Gln Ser Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410
415 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Val Arg Gly
420 425 430 Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn
Lys Ala 435 440 445 Leu Asn Asp Leu Cys Ile Arg Val Asn Asn Ser Glu
Leu Phe Phe Val 450 455 460 Ala Ser Glu Ser Ser Tyr Asn Glu Asn Asp
Ile Asn Thr Pro Lys Glu465 470 475 480 Ile Asp Asp Thr Thr Asn Leu
Asn Asn Asn Tyr Arg Asn Asn Leu Asp 485 490 495 Glu Val Ile Leu Asp
Tyr Asn Ser Gln Thr Ile Pro Gln Ile Ser Asn 500 505 510 Arg Thr Leu
Asn Thr Leu Val Gln Asp Asn Ser Tyr Val Pro Arg Tyr 515 520 525 Asp
Ser Asn Gly Thr Ser Glu Ile Glu Glu Tyr Asp Val Val Asp Phe 530 535
540 Asn Val Phe Phe Tyr Leu His Ala Gln Lys Val Pro Glu Gly Glu
Thr545 550 555 560 Asn Ile Ser Leu Thr Ser Ser Ile Asp Thr Ala Leu
Leu Glu Glu Ser 565 570 575 Lys Asp Ile Phe Phe Ser Ser Glu Phe Ile
Asp Thr Ile Asn Lys Pro 580 585 590 Val Asn Ala Ala Leu Phe Ile Asp
Trp Ile Ser Lys Val Ile Arg Asp 595 600 605 Phe Thr Thr Glu Ala Thr
Gln Lys Ser Thr Val Asp Lys Ile Ala Asp 610 615 620 Ile Ser Leu Ile
Val Pro Tyr Val Gly Leu Ala Leu Asn Ile Ile Ile625 630 635 640 Glu
Ala Glu Lys Gly Asn Phe Glu Glu Ala Phe Glu Leu Leu Gly Val 645 650
655 Gly Ile Leu Leu Glu Phe Val Pro Glu Leu Thr Ile Pro Val Ile Leu
660 665 670 Val Phe Thr Ile Lys Ser Tyr Ile Asp Ser Tyr Glu Asn Lys
Asn Lys 675 680 685 Ala Ile Lys Ala Ile Asn Asn Ser Leu Ile Glu Arg
Glu Ala Lys Trp 690 695 700 Lys Glu Ile Tyr Ser Trp Ile Val Ser Asn
Trp Leu Thr Arg Ile Asn705 710 715 720 Thr Gln Phe Asn Lys Arg Lys
Glu Gln Met Tyr Gln Ala Leu Gln Asn 725 730 735 Gln Val Asp Ala Ile
Lys Thr Ala Ile Glu Tyr Lys Tyr Asn Asn Tyr 740 745 750 Thr Ser Asp
Glu Lys Asn Arg Leu Glu Ser Glu Tyr Asn Ile Asn Asn 755 760 765 Ile
Glu Glu Glu Leu Asn Lys Lys Val Ser Leu Ala Met Lys Asn Ile 770 775
780 Glu Arg Phe Met Thr Glu Ser Ser Ile Ser Tyr Leu Met Lys Leu
Ile785 790 795 800 Asn Glu Ala Lys Val Gly Lys Leu Lys Lys Tyr Asp
Asn His Val Lys 805 810 815 Ser Asp Leu Leu Asn Tyr Ile Leu Asp His
Arg Ser Ile Leu Gly Glu 820 825 830 Gln Thr Asn Glu Leu Ser Asp Leu
Val Thr Ser Thr Leu Asn Ser Ser 835 840 845 Ile Pro Phe Glu Leu Ser
Ser Tyr Thr Asn Asp Lys Ile Leu Ile Ile 850 855 860 Tyr Phe Asn Arg
Leu Tyr Lys Lys Ile Lys Asp Ser Ser Ile Leu Asp865 870 875 880 Met
Arg Tyr Glu Asn Asn Lys Phe Ile Asp Ile Ser Gly Tyr Gly Ser 885 890
895 Asn Ile Ser Ile Asn Gly Asn Val Tyr Ile Tyr Ser Thr Asn Arg Asn
900 905 910 Gln Phe Gly Ile Tyr Asn Ser Arg Leu Ser Glu Val Asn Ile
Ala Gln 915 920 925 Asn Asn Asp Ile Ile Tyr Asn Ser Arg Tyr Gln Asn
Phe Ser Ile Ser 930 935 940 Phe Trp Val Arg Ile Pro Lys His Tyr Lys
Pro Met Asn His Asn Arg945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys
Met Gly Asn Asn Asn Ser Gly Trp Lys 965 970 975 Ile Ser Leu Arg Thr
Val Arg Asp Cys Glu Ile Ile Trp Thr Leu Gln 980 985 990 Asp Thr Ser
Gly Asn Lys Glu Asn Leu Ile Phe Arg Tyr Glu Glu Leu 995 1000 1005
Asn Arg Ile Ser Asn Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile Thr
1010 1015 1020 Asn Asn Arg Leu Gly Asn Ser Arg Ile Tyr Ile Asn Gly
Asn Leu Ile1025 1030 1035 1040Val Glu Lys Ser Ile Ser Asn Leu Gly
Asp Ile His Val Ser Asp Asn 1045 1050 1055 Ile Leu Phe Lys Ile Val
Gly Cys Asp Asp Glu Thr Tyr Val Gly Ile 1060 1065 1070 Arg Tyr Phe
Lys Val Phe Asn Thr Glu Leu Asp Lys Thr Glu Ile Glu 1075 1080 1085
Thr Leu Tyr Ser Asn Glu Pro Asp Pro Ser Ile Leu Lys Asn Tyr Trp
1090 1095 1100 Gly Asn Tyr Leu Leu Tyr Asn Lys Lys Tyr Tyr Leu Phe
Asn Leu Leu1105 1110 1115 1120Arg Lys Asp Lys Tyr Ile Thr Leu Asn
Ser Gly Ile Leu Asn Ile Asn 1125 1130 1135 Gln Gln Arg Gly Val Thr
Glu Gly Ser Val Phe Leu Asn Tyr Lys Leu 1140 1145 1150 Tyr Glu Gly
Val Glu Val Ile Ile Arg Lys Asn Gly Pro Ile Asp Ile 1155 1160 1165
Ser Asn Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala Tyr Ile Asn
1170 1175 1180 Val Val Asp Arg Gly Val Glu Tyr Arg Leu Tyr Ala Asp
Thr Lys Ser1185 1190 1195 1200Glu Lys Glu Lys Ile Ile Arg Thr Ser
Asn Leu Asn Asp Ser Leu Gly 1205 1210 1215 Gln Ile Ile Val Met Asp
Ser Ile Gly Asn Asn Cys Thr Met Asn Phe 1220 1225 1230 Gln Asn Asn
Asn Gly Ser Asn Ile Gly Leu Leu Gly Phe His Ser Asn 1235 1240 1245
Asn Leu Val Ala Ser Ser Trp Tyr Tyr Asn Asn Ile Arg Arg Asn Thr
1250 1255 1260 Ser Ser Asn Gly Cys Phe Trp Ser Ser Ile Ser Lys Glu
Asn Gly Trp1265 1270 1275 1280Lys Glu551307PRTArtificial
SequenceBoNT/G-DiA 55Met Pro Val Asn Ile Lys Asn Phe Asn Tyr Asn
Asp Pro Ile Asn Asn1 5 10 15 Asp Asp Ile Ile Met Met Glu Pro Phe
Asn Asp Pro Gly Pro Gly Thr 20 25 30 Tyr Tyr Lys Ala Phe Arg Ile
Ile Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Phe Thr Tyr Gly
Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly 50 55 60 Val Phe Ser
Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys65 70 75 80 Thr
Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 85 90
95 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile
100 105 110 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro
Asp Lys 115 120 125 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys
Lys Ile Ile Gln 130 135 140 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu
Met Thr Asn Leu Ile Ile145 150 155 160 Phe Gly Pro Gly Pro Val Leu
Ser Asp Asn Phe Thr Asp Ser Met Ile 165 170 175 Met Asn Gly His Ser
Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190 Ile Arg Phe
Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu 195 200 205 Asn
Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215
220 Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu
Tyr225 230 235 240 Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn
Thr Lys Glu Phe 245 250 255 Phe Met Gln His Ser Asp Pro Val Gln Ala
Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly His Asp Pro Ser Val Ile
Ser Pro Ser Thr Asp Met Asn Ile 275 280 285 Tyr Asn Lys Ala Leu Gln
Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290 295 300 Ile Val Ser Ser
Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys305 310 315 320 Gln
Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330
335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met
340 345 350 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile
Lys Thr 355 360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile
Lys Thr Glu Lys 370 375 380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn
Glu Gly Phe Asn Ile Ala385 390 395 400 Ser Lys Asn Leu Lys Thr Glu
Phe Asn Gly Gln Asn Lys Ala Val Asn 405 410 415 Lys Glu Ala Tyr Glu
Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg 420 425 430 Ile Ala Met
Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu 435 440 445 Asp
Lys Gly Tyr Asn Lys Ala Leu Asn Asp Leu Cys Ile Ile Val Asn 450 455
460 Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys Asp Ser Phe Ser Lys
Asp465 470 475 480 Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn Thr Gln
Asn Asn Thr Ile 485 490 495 Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile
Leu Asp Asn Asp Leu Ser 500 505 510 Ser Gly Ile Asp Leu Pro Asn Glu
Asn Thr Glu Pro Phe Thr Asn Phe 515 520 525 Asp Asp Ile Asp Ile Pro
Val Tyr Ile Lys Gln Ser Ala Leu Lys Lys 530 535 540 Ile Phe Val Asp
Gly Asp Ser Leu Phe Glu Tyr Leu His Ala Gln Thr545 550 555 560 Phe
Pro Ser Asn Ile Glu Asn Leu Gln Leu Thr Asn Ser Leu Asn Asp 565 570
575 Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr Phe Phe Ser Thr Asn Leu
580 585 590 Val Glu Lys Ala Asn Thr Val Val Gly Ala Ser Leu Phe Val
Asn Trp 595 600 605 Val Lys Gly Val Ile Asp Asp Phe Thr Ser Glu Ser
Thr Gln Lys Ser 610 615 620 Thr Ile Asp Lys Val Ser Asp Val Ser Ile
Ile Ile Pro Tyr Ile Gly625 630 635 640 Pro Ala Leu Asn Val Gly Asn
Glu Thr Ala Lys Glu Asn Phe Lys Asn 645 650 655 Ala Phe Glu Ile Gly
Gly Ala Ala Ile Leu Met Glu Phe Ile Pro Glu 660 665 670 Leu Ile Val
Pro Ile Val Gly Phe Phe Thr Leu Glu Ser Tyr Val Gly 675 680 685 Asn
Lys Gly His Ile Ile Met Thr Ile Ser Asn Ala Leu Lys Lys Arg 690 695
700 Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu Ile Val Ser Gln Trp
Leu705 710 715 720 Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu
Arg Met Tyr Asn 725 730 735 Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu
Lys Ile Ile Glu Asp Gln 740 745 750 Tyr Asn Arg Tyr Ser Glu Glu Asp
Lys Met Asn Ile Asn Ile Asp Phe 755 760 765 Asn Asp Ile Asp Phe Lys
Leu Asn Gln Ser Ile Asn Leu Ala Ile Asn 770 775 780 Asn Ile Asp Asp
Phe Ile Asn Gln Cys Ser Ile Ser Tyr Leu Met Asn785 790 795 800 Arg
Met Ile Pro Leu Ala Val Lys Lys Leu Lys Asp Phe Asp Asp Asn 805 810
815 Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp Thr Asn Glu Leu Tyr Leu
820 825 830 Leu Asp Glu Val Asn Ile Leu Lys Ser Lys Val Asn Arg His
Leu Lys 835 840 845 Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr Thr Lys
Asp Thr Ile Leu 850 855 860 Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn
Ile Ser Ser Asn Ala Ile865 870 875 880 Leu Ser Leu Ser Tyr Arg Gly
Gly Arg Leu Ile Asp Ser Ser Gly Tyr 885 890 895 Gly Ala Thr Met Asn
Val Gly Ser Asp Val Ile Phe Asn Asp Ile Gly 900 905 910 Asn Gly Gln
Phe Lys Leu Asn Asn Ser Glu Asn Ser Asn Ile Thr Ala 915 920 925 His
Gln Ser Lys Phe Val Val Tyr Asp Ser Met Phe Asp Asn Phe Ser 930 935
940 Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr Asn Asn Asn Asp Ile
Gln945 950 955 960 Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile Ser Cys
Ile Lys Asn Asp 965 970 975 Ser Gly Trp Lys Val Ser Ile Lys Gly Asn
Arg Ile Ile Trp Thr Leu 980 985 990 Ile Asp Val Asn Ala Lys Ser Lys
Ser Ile Phe Phe Glu Tyr Ser Ile 995 1000 1005 Lys Asp Asn Ile Ser
Asp Tyr Ile Asn Lys Trp Phe Ser Ile Thr Ile 1010 1015 1020 Thr Asn
Asp Arg Leu Gly Asn Ala Asn Ile Tyr Ile Asn
Gly Ser Leu1025 1030 1035 1040Lys Lys Ser Glu Lys Ile Leu Asn Leu
Asp Arg Ile Asn Ser Ser Asn 1045 1050 1055 Asp Ile Asp Phe Lys Leu
Ile Asn Cys Thr Asp Thr Thr Lys Phe Val 1060 1065 1070 Trp Ile Lys
Asp Phe Asn Ile Phe Gly Arg Glu Leu Asn Ala Thr Glu 1075 1080 1085
Val Ser Ser Leu Tyr Trp Ile Gln Ser Ser Thr Asn Thr Leu Lys Asp
1090 1095 1100 Phe Trp Gly Asn Pro Leu Arg Tyr Asp Thr Gln Tyr Tyr
Leu Phe Asn1105 1110 1115 1120Gln Gly Met Gln Asn Ile Tyr Ile Lys
Tyr Phe Ser Lys Ala Ser Met 1125 1130 1135 Gly Glu Thr Ala Pro Arg
Thr Asn Phe Asn Asn Ala Ala Ile Asn Tyr 1140 1145 1150 Gln Asn Leu
Tyr Leu Gly Leu Arg Phe Ile Ile Lys Lys Ala Ser Asn 1155 1160 1165
Ser Arg Asn Ile Asn Asn Asp Asn Ile Val Arg Glu Gly Asp Tyr Ile
1170 1175 1180 Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu Ser Tyr Arg
Val Tyr Val1185 1190 1195 1200Leu Val Asn Ser Lys Glu Ile Gln Thr
Gln Leu Phe Leu Ala Pro Ile 1205 1210 1215 Asn Asp Asp Pro Thr Phe
Tyr Asp Val Leu Gln Ile Lys Lys Tyr Tyr 1220 1225 1230 Glu Lys Thr
Thr Tyr Asn Cys Gln Ile Leu Cys Glu Lys Asp Thr Lys 1235 1240 1245
Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe Val Lys Asp Tyr Gly Tyr
1250 1255 1260 Val Trp Asp Thr Tyr Asp Asn Tyr Phe Cys Ile Ser Gln
Trp Tyr Leu1265 1270 1275 1280Arg Arg Ile Ser Glu Asn Ile Asn Lys
Leu Arg Leu Gly Cys Asn Trp 1285 1290 1295 Gln Phe Ile Pro Val Asp
Glu Gly Trp Thr Glu 1300 1305 561311PRTArtificial SequenceTeNT-DiA
56Met Pro Ile Thr Ile Asn Asn Phe Arg Tyr Ser Asp Pro Val Asn Asn1
5 10 15 Asp Thr Ile Ile Met Met Glu Pro Pro Tyr Cys Lys Gly Leu Asp
Ile 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile
Val Pro Glu 35 40 45 Arg Tyr Glu Phe Gly Thr Lys Pro Glu Asp Phe
Asn Pro Pro Ser Ser 50 55 60 Leu Ile Glu Gly Ala Ser Glu Tyr Tyr
Asp Pro Asn Tyr Leu Arg Thr65 70 75 80 Asp Ser Asp Lys Asp Arg Phe
Leu Gln Thr Met Val Lys Leu Phe Asn 85 90 95 Arg Ile Lys Asn Asn
Val Ala Gly Glu Ala Leu Leu Asp Lys Ile Ile 100 105 110 Asn Ala Ile
Pro Tyr Leu Gly Asn Ser Tyr Ser Leu Leu Asp Lys Phe 115 120 125 Asp
Thr Asn Ser Asn Ser Val Ser Phe Asn Leu Leu Glu Gln Asp Pro 130 135
140 Ser Gly Ala Thr Thr Lys Ser Ala Met Leu Thr Asn Leu Ile Ile
Phe145 150 155 160 Gly Pro Gly Pro Val Leu Asn Lys Asn Glu Val Arg
Gly Ile Val Leu 165 170 175 Arg Val Asp Asn Lys Asn Tyr Phe Pro Cys
Arg Asp Gly Phe Gly Ser 180 185 190 Ile Met Gln Met Ala Phe Cys Pro
Glu Tyr Val Pro Thr Phe Asp Asn 195 200 205 Val Ile Glu Asn Ile Thr
Ser Leu Thr Ile Gly Lys Ser Lys Tyr Phe 210 215 220 Gln Asp Pro Ala
Leu Leu Leu Met His Glu Leu Ile His Val Leu His225 230 235 240 Gly
Leu Tyr Gly Met Gln Val Ser Ser His Glu Ile Ile Pro Ser Lys 245 250
255 Gln Glu Ile Tyr Met Gln His Thr Tyr Pro Ile Ser Ala Glu Glu Leu
260 265 270 Phe Thr Phe Gly Gly Gln Asp Ala Asn Leu Ile Ser Ile Asp
Ile Lys 275 280 285 Asn Asp Leu Tyr Glu Lys Thr Leu Asn Asp Tyr Lys
Ala Ile Ala Asn 290 295 300 Lys Leu Ser Gln Val Thr Ser Cys Asn Asp
Pro Asn Ile Asp Ile Asp305 310 315 320 Ser Tyr Lys Gln Ile Tyr Gln
Gln Lys Tyr Gln Phe Asp Lys Asp Ser 325 330 335 Asn Gly Gln Tyr Ile
Val Asn Glu Asp Lys Phe Gln Ile Leu Tyr Asn 340 345 350 Ser Ile Met
Tyr Gly Phe Thr Glu Ile Glu Leu Gly Lys Lys Phe Asn 355 360 365 Ile
Lys Thr Arg Leu Ser Tyr Phe Ser Met Asn His Asp Pro Val Lys 370 375
380 Ile Pro Asn Leu Leu Asp Asp Thr Ile Tyr Asn Asp Thr Glu Gly
Phe385 390 395 400 Asn Ile Glu Ser Lys Asp Leu Lys Ser Glu Tyr Lys
Gly Gln Asn Met 405 410 415 Arg Val Asn Thr Asn Ala Phe Arg Asn Val
Asp Gly Ser Gly Leu Val 420 425 430 Ser Lys Leu Ile Gly Leu Cys Val
Arg Gly Ile Ile Thr Ser Lys Thr 435 440 445 Lys Ser Leu Asp Lys Gly
Tyr Asn Lys Ala Leu Asn Asp Leu Cys Ile 450 455 460 Lys Ile Lys Asn
Glu Asp Leu Thr Phe Ile Ala Glu Lys Asn Ser Phe465 470 475 480 Ser
Glu Glu Pro Phe Gln Asp Glu Ile Val Ser Tyr Asn Thr Lys Asn 485 490
495 Lys Pro Leu Asn Phe Asn Tyr Ser Leu Asp Lys Ile Ile Val Asp Tyr
500 505 510 Asn Leu Gln Ser Lys Ile Thr Leu Pro Asn Asp Arg Thr Thr
Pro Val 515 520 525 Thr Lys Gly Ile Pro Tyr Ala Pro Glu Tyr Lys Ser
Asn Ala Ala Ser 530 535 540 Thr Ile Glu Ile His Asn Ile Asp Asp Asn
Thr Ile Tyr Gln Tyr Leu545 550 555 560 Tyr Ala Gln Lys Ser Pro Thr
Thr Leu Gln Arg Ile Thr Met Thr Asn 565 570 575 Ser Val Asp Asp Ala
Leu Ile Asn Ser Thr Lys Ile Tyr Ser Tyr Phe 580 585 590 Pro Ser Val
Ile Ser Lys Val Asn Gln Gly Ala Gln Gly Ile Leu Phe 595 600 605 Leu
Gln Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser 610 615
620 Gln Lys Thr Thr Ile Asp Lys Ile Ser Asp Val Ser Thr Ile Val
Pro625 630 635 640 Tyr Ile Gly Pro Ala Leu Asn Ile Val Lys Gln Gly
Tyr Glu Gly Asn 645 650 655 Phe Ile Gly Ala Leu Glu Thr Thr Gly Val
Val Leu Leu Leu Glu Tyr 660 665 670 Ile Pro Glu Ile Thr Leu Pro Val
Ile Ala Ala Leu Ser Ile Ala Glu 675 680 685 Ser Ser Thr Gln Lys Glu
Lys Ile Ile Lys Thr Ile Asp Asn Phe Leu 690 695 700 Glu Lys Arg Tyr
Glu Lys Trp Ile Glu Val Tyr Lys Leu Val Lys Ala705 710 715 720 Lys
Trp Leu Gly Thr Val Asn Thr Gln Phe Gln Lys Arg Ser Tyr Gln 725 730
735 Met Tyr Arg Ser Leu Glu Tyr Gln Val Asp Ala Ile Lys Lys Ile Ile
740 745 750 Asp Tyr Glu Tyr Lys Ile Tyr Ser Gly Pro Asp Lys Glu Gln
Ile Ala 755 760 765 Asp Glu Ile Asn Asn Leu Lys Asn Lys Leu Glu Glu
Lys Ala Asn Lys 770 775 780 Ala Met Ile Asn Ile Asn Ile Phe Met Arg
Glu Ser Ser Arg Ser Phe785 790 795 800 Leu Val Asn Gln Met Ile Asn
Glu Ala Lys Lys Gln Leu Leu Glu Phe 805 810 815 Asp Thr Gln Ser Lys
Asn Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser 820 825 830 Lys Phe Ile
Gly Ile Thr Glu Leu Lys Lys Leu Glu Ser Lys Ile Asn 835 840 845 Lys
Val Phe Ser Thr Pro Ile Pro Phe Ser Tyr Ser Lys Asn Leu Asp 850 855
860 Cys Trp Val Asp Asn Glu Glu Asp Ile Asp Val Ile Leu Lys Lys
Ser865 870 875 880 Thr Ile Leu Asn Leu Asp Ile Asn Asn Asp Ile Ile
Ser Asp Ile Ser 885 890 895 Gly Phe Asn Ser Ser Val Ile Thr Tyr Pro
Asp Ala Gln Leu Val Pro 900 905 910 Gly Ile Asn Gly Lys Ala Ile His
Leu Val Asn Asn Glu Ser Ser Glu 915 920 925 Val Ile Val His Lys Ala
Met Asp Ile Glu Tyr Asn Asp Met Phe Asn 930 935 940 Asn Phe Thr Val
Ser Phe Trp Leu Arg Val Pro Lys Val Ser Ala Ser945 950 955 960 His
Leu Glu Gln Tyr Gly Thr Asn Glu Tyr Ser Ile Ile Ser Ser Met 965 970
975 Lys Lys His Ser Leu Ser Ile Gly Ser Gly Trp Ser Val Ser Leu Lys
980 985 990 Gly Asn Asn Leu Ile Trp Thr Leu Lys Asp Ser Ala Gly Glu
Val Arg 995 1000 1005 Gln Ile Thr Phe Arg Asp Leu Pro Asp Lys Phe
Asn Ala Tyr Leu Ala 1010 1015 1020 Asn Lys Trp Val Phe Ile Thr Ile
Thr Asn Asp Arg Leu Ser Ser Ala1025 1030 1035 1040Asn Leu Tyr Ile
Asn Gly Val Leu Met Gly Ser Ala Glu Ile Thr Gly 1045 1050 1055 Leu
Gly Ala Ile Arg Glu Asp Asn Asn Ile Thr Leu Lys Leu Asp Arg 1060
1065 1070 Cys Asn Asn Asn Asn Gln Tyr Val Ser Ile Asp Lys Phe Arg
Ile Phe 1075 1080 1085 Cys Lys Ala Leu Asn Pro Lys Glu Ile Glu Lys
Leu Tyr Thr Ser Tyr 1090 1095 1100 Leu Ser Ile Thr Phe Leu Arg Asp
Phe Trp Gly Asn Pro Leu Arg Tyr1105 1110 1115 1120Asp Thr Glu Tyr
Tyr Leu Ile Pro Val Ala Ser Ser Ser Lys Asp Val 1125 1130 1135 Gln
Leu Lys Asn Ile Thr Asp Tyr Met Tyr Leu Thr Asn Ala Pro Ser 1140
1145 1150 Tyr Thr Asn Gly Lys Leu Asn Ile Tyr Tyr Arg Arg Leu Tyr
Asn Gly 1155 1160 1165 Leu Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn
Asn Glu Ile Asp Ser 1170 1175 1180 Phe Val Lys Ser Gly Asp Phe Ile
Lys Leu Tyr Val Ser Tyr Asn Asn1185 1190 1195 1200Asn Glu His Ile
Val Gly Tyr Pro Lys Asp Gly Asn Ala Phe Asn Asn 1205 1210 1215 Leu
Asp Arg Ile Leu Arg Val Gly Tyr Asn Ala Pro Gly Ile Pro Leu 1220
1225 1230 Tyr Lys Lys Met Glu Ala Val Lys Leu Arg Asp Leu Lys Thr
Tyr Ser 1235 1240 1245 Val Gln Leu Lys Leu Tyr Asp Asp Lys Asn Ala
Ser Leu Gly Leu Val 1250 1255 1260 Gly Thr His Asn Gly Gln Ile Gly
Asn Asp Pro Asn Arg Asp Ile Leu1265 1270 1275 1280Ile Ala Ser Asn
Trp Tyr Phe Asn His Leu Lys Asp Lys Ile Leu Gly 1285 1290 1295 Cys
Asp Trp Tyr Phe Val Pro Thr Asp Glu Gly Trp Thr Asn Asp 1300 1305
1310 571278PRTArtificial SequenceBaNT-DiA 57Met Pro Val Asn Ile Asn
Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn1 5 10 15 Thr Thr Ile Leu
Tyr Met Lys Met Pro Tyr Tyr Glu Asp Ser Asn Lys 20 25 30 Tyr Tyr
Lys Ala Phe Glu Ile Met Asp Asn Val Trp Ile Ile Pro Glu 35 40 45
Arg Asn Ile Ile Gly Lys Lys Pro Ser Asp Phe Tyr Pro Pro Ile Ser 50
55 60 Leu Asp Ser Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr
Thr65 70 75 80 Asp Ala Glu Lys Asp Arg Phe Leu Lys Thr Val Ile Lys
Leu Phe Asn 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Gln Val Leu
Leu Glu Glu Ile Lys 100 105 110 Asn Gly Lys Pro Tyr Leu Gly Asn Asp
His Thr Ala Val Asn Glu Phe 115 120 125 Cys Ala Asn Asn Arg Ser Thr
Ser Val Glu Ile Lys Glu Ser Asn Gly 130 135 140 Thr Thr Asp Ser Met
Leu Leu Asn Leu Val Ile Leu Gly Pro Gly Pro145 150 155 160 Asn Ile
Leu Glu Cys Ser Thr Phe Pro Val Arg Ile Phe Pro Asn Asn 165 170 175
Ile Ala Tyr Asp Pro Ser Glu Lys Gly Phe Gly Ser Ile Gln Leu Met 180
185 190 Ser Phe Ser Thr Glu Tyr Glu Tyr Ala Phe Asn Asp Asn Thr Asp
Leu 195 200 205 Phe Ile Ala Asp Pro Ala Ile Ser Leu Ala His Glu Leu
Ile His Val 210 215 220 Leu His Gly Leu Tyr Gly Ala Lys Gly Val Thr
Asn Lys Lys Val Ile225 230 235 240 Glu Val Asp Gln Gly Ala Leu Met
Ala Ala Glu Lys Asp Ile Lys Ile 245 250 255 Glu Glu Phe Ile Thr Phe
Gly Gly Gln Asp Leu Asn Ile Ile Thr Asn 260 265 270 Ser Thr Asn Gln
Lys Ile Tyr Val Ile Leu Leu Ser Asn Tyr Thr Ala 275 280 285 Ile Ala
Ser Arg Leu Ser Gln Val Asn Arg Asn Asn Ser Ala Leu Asn 290 295 300
Thr Thr Tyr Tyr Lys Asn Phe Phe Gln Trp Lys Tyr Gly Leu Asp Gln305
310 315 320 Asp Ser Asn Gly Asn Tyr Thr Val Asn Ile Ser Lys Phe Asn
Ala Ile 325 330 335 Tyr Lys Lys Leu Phe Ser Phe Thr Glu Cys Asp Leu
Ala Gln Lys Phe 340 345 350 Gln Val Lys Asn Arg Ser Asn Tyr Leu Phe
His Phe Lys Pro Phe Arg 355 360 365 Leu Leu Asp Leu Leu Asp Asp Asn
Ile Tyr Ser Ile Ser Glu Gly Phe 370 375 380 Asn Ile Gly Ser Leu Arg
Val Asn Asn Asn Gly Gln Asn Ile Asn Leu385 390 395 400 Asn Ser Arg
Ile Val Gly Pro Ile Pro Asp Asn Gly Leu Val Glu Arg 405 410 415 Phe
Val Gly Leu Cys Val Arg Gly Ile Ile Thr Ser Lys Thr Lys Ser 420 425
430 Leu Asp Lys Gly Tyr Asn Lys Ala Leu Asn Asp Leu Cys Ile Lys Val
435 440 445 Asn Asn Arg Asp Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr
Asn Glu 450 455 460 Asn Gly Ile Asn Ser Pro Lys Glu Ile Asp Asp Thr
Thr Ile Thr Asn465 470 475 480 Asn Asn Tyr Lys Lys Asn Leu Asp Glu
Val Ile Leu Asp Tyr Asn Ser 485 490 495 Asp Ala Ile Pro Asn Leu Ser
Ser Arg Leu Leu Asn Thr Thr Ala Gln 500 505 510 Asn Asp Ser Tyr Val
Pro Lys Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525 Lys Glu Tyr
Thr Val Asp Lys Leu Asn Val Phe Phe Tyr Leu Tyr Ala 530 535 540 Gln
Lys Ala Pro Glu Gly Glu Ser Ala Ile Ser Leu Thr Ser Ser Val545 550
555 560 Asn Thr Ala Leu Leu Asp Ala Ser Lys Val Tyr Thr Phe Phe Ser
Ser 565 570 575 Asp Phe Ile Asn Thr Val Asn Lys Pro Val Gln Ala Ala
Leu Phe Ile 580 585 590 Ser Trp Ile Gln Gln Val Ile Asn Asp Phe Thr
Thr Glu Ala Thr Gln 595 600 605 Lys Ser Thr Ile Asp Lys Ile Ala Asp
Ile Ser Leu Ile Val Pro Tyr 610 615 620 Val Gly Leu Ala Leu Asn Ile
Gly Asn Glu Val Gln Lys Gly Asn Phe625 630 635 640 Lys Glu Ala Ile
Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Val 645 650 655 Pro Glu
Leu Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe 660 665 670
Ile Asn Ser Asp Asp Ser Lys Asn Lys Ile Ile Lys Ala Ile Asn Asn 675
680 685 Ala Leu Arg Glu Arg Glu Leu Lys Trp Lys Glu Val Tyr Ser Trp
Ile 690 695 700 Val Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn
Lys Arg Lys705 710
715 720 Glu Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp Gly Ile Lys
Lys 725 730 735 Ile Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Leu Asp Glu
Lys Asn Arg 740 745 750 Leu Arg Ala Glu Tyr Asn Ile Tyr Ser Ile Lys
Glu Glu Leu Asn Lys 755 760 765 Lys Val Ser Leu Ala Met Gln Asn Ile
Asp Arg Phe Leu Thr Glu Ser 770 775 780 Ser Ile Ser Tyr Leu Met Lys
Leu Ile Asn Glu Ala Lys Ile Asn Lys785 790 795 800 Leu Ser Glu Tyr
Asp Lys Arg Val Asn Gln Tyr Leu Leu Asn Tyr Ile 805 810 815 Leu Glu
Asn Ser Ser Thr Leu Gly Thr Ser Ser Val Pro Glu Leu Asn 820 825 830
Asn Leu Val Ser Asn Thr Leu Asn Asn Ser Ile Pro Phe Glu Leu Ser 835
840 845 Glu Tyr Thr Asn Asp Lys Ile Leu Ile His Ile Leu Ile Arg Phe
Tyr 850 855 860 Lys Arg Ile Ile Asp Ser Ser Ile Leu Asn Met Lys Tyr
Glu Asn Asn865 870 875 880 Arg Phe Ile Asp Ser Ser Gly Tyr Gly Ser
Asn Ile Ser Ile Asn Gly 885 890 895 Asp Ile Tyr Ile Tyr Ser Thr Asn
Arg Asn Gln Phe Gly Ile Tyr Ser 900 905 910 Ser Arg Leu Ser Glu Val
Asn Ile Thr Gln Asn Asn Thr Ile Ile Tyr 915 920 925 Asn Ser Arg Tyr
Gln Asn Phe Ser Val Ser Phe Trp Val Arg Ile Pro 930 935 940 Lys Tyr
Asn Asn Leu Lys Asn Leu Asn Asn Glu Tyr Thr Ile Ile Asn945 950 955
960 Cys Met Arg Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Asn Tyr Asn
965 970 975 Asn Ile Ile Trp Thr Leu Gln Asp Thr Thr Gly Asn Asn Gln
Lys Leu 980 985 990 Val Phe Asn Tyr Thr Gln Met Ile Asp Ile Ser Asp
Tyr Ile Asn Lys 995 1000 1005 Trp Thr Phe Val Thr Ile Thr Asn Asn
Arg Leu Gly His Ser Lys Leu 1010 1015 1020 Tyr Ile Asn Gly Asn Leu
Thr Asp Gln Lys Ser Ile Leu Asn Leu Gly1025 1030 1035 1040Asn Ile
His Val Asp Asp Asn Ile Leu Phe Lys Ile Val Gly Cys Asn 1045 1050
1055 Asp Thr Arg Tyr Val Gly Ile Arg Tyr Phe Lys Ile Phe Asn Met
Glu 1060 1065 1070 Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr His Ser
Glu Pro Asp Ser 1075 1080 1085 Thr Ile Leu Lys Asp Phe Trp Gly Asn
Tyr Leu Leu Tyr Asn Lys Lys 1090 1095 1100 Tyr Tyr Leu Leu Asn Leu
Leu Lys Pro Asn Met Ser Val Thr Lys Asn1105 1110 1115 1120Ser Asp
Ile Leu Asn Ile Asn Arg Gln Arg Gly Ile Tyr Ser Lys Thr 1125 1130
1135 Asn Ile Phe Ser Asn Ala Arg Leu Tyr Thr Gly Val Glu Val Ile
Ile 1140 1145 1150 Arg Lys Val Gly Ser Thr Asp Thr Ser Asn Thr Asp
Asn Phe Val Arg 1155 1160 1165 Lys Asn Asp Thr Val Tyr Ile Asn Val
Val Asp Gly Asn Ser Glu Tyr 1170 1175 1180 Gln Leu Tyr Ala Asp Val
Ser Thr Ser Ala Val Glu Lys Thr Ile Lys1185 1190 1195 1200Leu Arg
Arg Ile Ser Asn Ser Asn Tyr Asn Ser Asn Gln Met Ile Ile 1205 1210
1215 Met Asp Ser Ile Gly Asp Asn Cys Thr Met Asn Phe Lys Thr Asn
Asn 1220 1225 1230 Gly Asn Asp Ile Gly Leu Leu Gly Phe His Leu Asn
Asn Leu Val Ala 1235 1240 1245 Ser Ser Trp Tyr Tyr Lys Asn Ile Arg
Asn Asn Thr Arg Asn Asn Gly 1250 1255 1260 Cys Phe Trp Ser Phe Ile
Ser Lys Glu His Gly Trp Gln Glu1265 1270 1275 581261PRTArtificial
SequenceBuNT-DiA 58Met Pro Thr Ile Asn Ser Phe Asn Tyr Asn Asp Pro
Val Asn Asn Arg1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys
Gln Gln Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp
Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Ile Pro Gln Asp
Phe Leu Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr
Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Gln Glu Lys65 70 75 80 Asp Lys
Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asp 85 90 95
Asn Leu Ser Gly Arg Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100
105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Gly Asp Phe Ile Ile Asn
Asp 115 120 125 Ala Ser Ala Val Pro Ile Gln Phe Ser Asn Gly Ser Gln
Ser Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro
Asp Leu Phe Glu Thr145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg
Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly Ser Ile Ala
Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe Lys Asp
Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr Leu
Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220
Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu225
230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr
Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser
Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile
Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Val Ser Asn Pro Leu Leu
Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp
Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn305 310 315 320 Ile Asn Lys
Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe
Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345
350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile
355 360 365 Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val
Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile
Thr Pro Ile Thr385 390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile
Arg Phe Cys Val Arg Gly Ile 405 410 415 Ile Thr Ser Lys Thr Lys Ser
Leu Asp Lys Gly Tyr Asn Lys Ala Leu 420 425 430 Asn Asp Leu Cys Ile
Glu Ile Asn Asn Gly Glu Leu Phe Phe Val Ala 435 440 445 Ser Glu Asn
Ser Tyr Asn Asp Asp Asn Ile Asn Thr Pro Lys Glu Ile 450 455 460 Asp
Asp Thr Val Thr Ser Asn Asn Asn Tyr Glu Asn Asp Leu Asp Gln465 470
475 480 Val Ile Leu Asn Phe Asn Ser Glu Ser Ala Pro Gly Leu Ser Asp
Glu 485 490 495 Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala Tyr Ile Pro
Lys Tyr Asp 500 505 510 Ser Asn Gly Thr Ser Asp Ile Glu Gln His Asp
Val Asn Glu Leu Asn 515 520 525 Val Phe Phe Tyr Leu Asp Ala Gln Lys
Val Pro Glu Gly Glu Asn Asn 530 535 540 Val Asn Leu Thr Ser Ser Ile
Asp Thr Ala Leu Leu Glu Gln Pro Lys545 550 555 560 Ile Tyr Thr Phe
Phe Ser Ser Glu Phe Ile Asn Asn Val Asn Lys Pro 565 570 575 Val Gln
Ala Ala Leu Phe Val Gly Trp Ile Gln Gln Val Leu Val Asp 580 585 590
Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr Val Asp Lys Ile Ala Asp 595
600 605 Ile Ser Ile Val Val Pro Tyr Ile Gly Leu Ala Leu Asn Ile Gly
Asn 610 615 620 Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala Leu Glu Leu
Leu Gly Ala625 630 635 640 Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu
Leu Ile Pro Thr Ile Leu 645 650 655 Val Phe Thr Ile Lys Ser Phe Leu
Gly Ser Ser Asp Asn Lys Asn Lys 660 665 670 Val Ile Lys Ala Ile Asn
Asn Ala Leu Lys Glu Arg Asp Glu Lys Trp 675 680 685 Lys Glu Val Tyr
Ser Phe Ile Val Ser Asn Trp Met Thr Lys Ile Asn 690 695 700 Thr Gln
Phe Asn Lys Arg Lys Glu Gln Met Tyr Gln Ala Leu Gln Asn705 710 715
720 Gln Val Asn Ala Leu Lys Ala Ile Ile Glu Ser Lys Tyr Asn Ser Tyr
725 730 735 Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn Lys Tyr Asp Ile
Glu Gln 740 745 750 Ile Glu Asn Glu Leu Asn Gln Lys Val Ser Ile Ala
Met Asn Asn Ile 755 760 765 Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser
Tyr Leu Met Lys Leu Ile 770 775 780 Asn Glu Val Lys Ile Asn Lys Leu
Arg Glu Tyr Asp Glu Asn Val Lys785 790 795 800 Thr Tyr Leu Leu Asp
Tyr Ile Ile Lys His Gly Ser Ile Leu Gly Glu 805 810 815 Ser Gln Gln
Glu Leu Asn Ser Met Val Ile Asp Thr Leu Asn Asn Ser 820 825 830 Ile
Pro Phe Lys Leu Ser Ser Tyr Thr Asp Asp Lys Ile Leu Ile Ser 835 840
845 Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys Ser Ser Ser Val Leu Asn
850 855 860 Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp Thr Ser Gly Tyr
Asp Ser865 870 875 880 Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys Tyr
Pro Thr Asn Lys Asn 885 890 895 Gln Phe Gly Ile Tyr Asn Asp Lys Leu
Ser Glu Val Asn Ile Ser Gln 900 905 910 Asn Asp Tyr Ile Ile Tyr Asp
Asn Lys Tyr Lys Asn Phe Ser Ile Ser 915 920 925 Phe Trp Val Arg Ile
Pro Asn Tyr Asp Asn Lys Ile Val Asn Val Asn 930 935 940 Asn Glu Tyr
Thr Ile Ile Asn Cys Met Arg Asp Asn Asn Ser Gly Trp945 950 955 960
Lys Val Ser Leu Asn His Asn Glu Ile Ile Trp Thr Leu Gln Asp Asn 965
970 975 Ser Gly Ile Asn Gln Lys Leu Ala Phe Asn Tyr Gly Asn Ala Asn
Gly 980 985 990 Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe Val Thr Ile
Thr Asn Asp 995 1000 1005 Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn
Gly Asn Leu Ile Asp Lys 1010 1015 1020 Lys Ser Ile Leu Asn Leu Gly
Asn Ile His Val Ser Asp Asn Ile Leu1025 1030 1035 1040Phe Lys Ile
Val Asn Cys Ser Tyr Thr Arg Tyr Ile Gly Ile Arg Tyr 1045 1050 1055
Phe Asn Ile Phe Asp Lys Glu Leu Asp Glu Thr Glu Ile Gln Thr Leu
1060 1065 1070 Tyr Asn Asn Glu Pro Asn Ala Asn Ile Leu Lys Asp Phe
Trp Gly Asn 1075 1080 1085 Tyr Leu Leu Tyr Asp Lys Glu Tyr Tyr Leu
Leu Asn Val Leu Lys Pro 1090 1095 1100 Asn Asn Phe Ile Asn Arg Arg
Thr Asp Ser Thr Leu Ser Ile Asn Asn1105 1110 1115 1120Ile Arg Ser
Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser Gly Ile Lys 1125 1130 1135
Val Lys Ile Gln Arg Val Asn Asn Ser Ser Thr Asn Asp Asn Leu Val
1140 1145 1150 Arg Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala Ser
Lys Thr His 1155 1160 1165 Leu Leu Pro Leu Tyr Ala Asp Thr Ala Thr
Thr Asn Lys Glu Lys Thr 1170 1175 1180 Ile Lys Ile Ser Ser Ser Gly
Asn Arg Phe Asn Gln Val Val Val Met1185 1190 1195 1200Asn Ser Val
Gly Asn Cys Thr Met Asn Phe Lys Asn Asn Asn Gly Asn 1205 1210 1215
Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr Val Val Ala Ser Thr
1220 1225 1230 Trp Tyr Tyr Thr His Met Arg Asp Asn Thr Asn Ser Asn
Gly Phe Phe 1235 1240 1245 Trp Asn Phe Ile Ser Glu Glu His Gly Trp
Gln Glu Lys 1250 1255 1260
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