U.S. patent application number 13/926660 was filed with the patent office on 2013-10-17 for methods for the delivery of toxins or enzymatically active portions thereof.
The applicant listed for this patent is Trustees of Tufts College. Invention is credited to Chuehling Kuo, George A. Oyler, Charles B. Shoemaker.
Application Number | 20130273590 13/926660 |
Document ID | / |
Family ID | 42077580 |
Filed Date | 2013-10-17 |
United States Patent
Application |
20130273590 |
Kind Code |
A1 |
Oyler; George A. ; et
al. |
October 17, 2013 |
Methods for the delivery of toxins or enzymatically active portions
thereof
Abstract
The present invention relates to methods, systems, and kits for
intoxicating cells, neuronal and non-neuronal cells, with a toxin
or fragment thereof. This is done by subjecting toxin substrate and
a lipid or polymeric carrier (e.g., DNA uptake facilitating agent)
to one or more cells for use in cell based assays. In an aspect,
the methods of the present invention allow for high throughput
assays and, as such, for the evaluation of drug candidates.
Inventors: |
Oyler; George A.;
(Baltimore, MD) ; Shoemaker; Charles B.; (North
Grafton, MA) ; Kuo; Chuehling; (Framingham,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trustees of Tufts College |
Medford |
MA |
US |
|
|
Family ID: |
42077580 |
Appl. No.: |
13/926660 |
Filed: |
June 25, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12690427 |
Jan 20, 2010 |
8492109 |
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13926660 |
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61145820 |
Jan 20, 2009 |
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Current U.S.
Class: |
435/29 ; 435/219;
435/325; 435/352; 435/369; 435/375; 435/441 |
Current CPC
Class: |
C12N 5/0686 20130101;
G01N 2333/415 20130101; G01N 2333/28 20130101; G01N 2333/33
20130101; C12N 9/52 20130101; G01N 2333/21 20130101; C12Y 304/24069
20130101; C12N 5/0618 20130101; G01N 2333/43517 20130101; G01N
2333/705 20130101; G01N 2333/25 20130101; G01N 33/5014 20130101;
G01N 2333/32 20130101 |
Class at
Publication: |
435/29 ; 435/375;
435/441; 435/219; 435/325; 435/369; 435/352 |
International
Class: |
C12N 5/079 20060101
C12N005/079; C12N 5/071 20060101 C12N005/071 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
AI030050 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method of intoxicating a cell with a toxin or an enzymatically
active fragment thereof, the method comprising: a. mixing the toxin
or the enzymatically active fragment thereof with a lipid or
polymeric carrier, thereby forming a mixture; and b. exposing the
mixture to one or more cells; wherein the cell becomes intoxicated
with said toxin or said enzymatically active fragment.
2. The method of claim 1, wherein the toxin or fragment thereof
comprises at least an enzymatic portion of botulinum neurotoxin,
tetanospasmin, tetrodotoxin, Clostridium difficile toxin Tcd A,
Clostridium difficile toxin Tcd B, Clostridium Lethal Toxin,
Anthrax Lethal Factor, Anthrax Edema Factor, Ricin, Exotoxin A,
Diphtheria toxin, Cholera toxin, Tetanus toxins, Shiga toxin,
latrotoxin, or a combination thereof.
3. The method of claim 2, wherein the enzymatically active fragment
of the toxin comprises the light chain or chain A of said
toxin.
4. The method of claim 3, wherein the light chain or chain A
includes a mutation or deletion.
5. The method of claim 4, wherein the botulinum neurotoxin or light
chain portion is derived from a serotype with a sequence similarity
of at least about 40% to a sequence, wherein the serotype is
selected from the group consisting of serotype A, B, C, D, E, F,
and G, and wherein the sequence is selected from the group
consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and
22.
6. The method of claim 1, wherein the lipid or polymeric carrier
comprises one or more lipid or polymeric carrier type DNA
transfection reagents.
7. The method of claim 6, wherein the DNA transfection reagent
comprises polylysine, polyethylenimine (PEI), a polymeric carrier,
a cationic lipid reagent, polycationic polymers, or any combination
thereof.
8. The method of claim 6, wherein the DNA transfection reagent is
in an amount between about 0.1 pM and about 1 .mu.M.
9. The method of claim 8, wherein the DNA transfection reagent is
in an amount between about 1 nM and about 10 nM.
10. The method of claim 6, wherein the toxin or the enzymatically
active fragment thereof is mixed with the DNA transfection reagent
for a length of time between about 5 minutes and about 72
hours.
11. The method of claim 10, wherein the toxin or the enzymatically
active fragment thereof is mixed with the DNA transfection reagent
for a length of time between about 1 hour and about 6 hours.
12. A method of delivering a botulinum neurotoxin or an
enzymatically active fragment thereof to the inside of one or more
cells in vitro, the method comprising: a. contacting the botulinum
neurotoxin or an enzymatically active fragment of the toxin with a
lipid or polymeric carrier to thereby obtain a mixture; and b.
exposing the one or more cells to the mixture in an amount to allow
the botulinum neurotoxin or enzymatically active fragment thereof
to enter the cell; wherein the cells are intoxicated with the
botulinum neurotoxin or the enzymatically active fragment
thereof.
13. The method of claim 12, wherein the botulinum neurotoxin
comprises a serotype selected from the group consisting of serotype
A, B, C, D, E, F, and G.
14. The method of claim 12, wherein the enzymatically active
fragment comprises the light chain or portion thereof from a
serotype with a sequence similarity of at least about 40% to a
sequence, wherein the serotype is selected from the group
consisting of serotype A, B, C, D, E, F, and G, and wherein the
sequence is selected from the group consisting of SEQ ID NO: 2, 4,
6, 8, 10, 12, 14, 16, 18, 20, and 22.
15. The method of claim 12, further including assessing the level
of one or more SNARE proteins to determine a level of cell
intoxication, wherein the SNARE proteins comprise synaptobrevin 2,
syntaxin, SNAP 25, or a combination thereof.
16. A method of preparing one or more cells for a cell-based assay,
wherein the cell is intoxicated with a toxin or an enzymatically
active fragment thereof, the method comprising: a. contacting the
botulinum neurotoxin or an enzymatically active fragment of the
toxin with a lipid or polymeric carrier to thereby obtain a
mixture; and b. exposing the one or more cells to the mixture in an
amount to allow the botulinum neurotoxin or the enzymatically
active fragment thereof to enter the one or more cells; wherein the
one or more cells are intoxicated with the toxin or the
enzymatically active fragment thereof.
17. A system or kit for delivering a toxin, an enzymatically active
fragment thereof, or a recombinant SNARE endoprotease to inside of
a cell in vitro, wherein the system or kit comprises: a. one or
more toxins, enzymatically active fragments thereof, or recombinant
SNARE endoproteases; and b. one or more lipid or polymeric
carriers.
18. The system or kit of claim 17, further including one or more
cells.
19. The system or kit of claim 18, wherein the one or more cells
are transfected with a nucleic acid molecule having a sequence that
comprises SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or
combination thereof.
20. An isolated cell that comprises a toxin, an enzymatically
active fragment thereof, or a recombinant SNARE endoprotease,
wherein the cell comprises HEK293, HIT-T15, a neuroendocrine
derived cell line, an immortalized cell line, or a tumor derived
cell line.
Description
RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 12/690,427, filed Jan. 20, 2010, which claims the benefit of
U.S. Provisional Application No. 61/145,820, filed Jan. 20, 2009,
entitled "Methods For The Delivery Of Toxins Or Enzymatically
Active Portions Thereof" by Oyler, George A. et al. The entire
teachings of the above applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] Prior to the present invention, cell-based assays for toxins
such as Botulinum neurotoxins (BoNT) are limited to cells that
contain the appropriate surface receptors used by the specific
toxin to gain entry into cells. The efficiency of toxin entry into
immortalized cell lines is often much poorer than that obtained
with primary cultures, requiring the use of high concentrations of
toxin and acceptance that a low percentage of the immortalized
cells will become intoxicated. The use of holotoxin, the native
toxin, in screening adds a substantial complication due to
additional regulatory, safety and waste disposal issues.
[0004] Also, cell-based assays for toxins can be important in the
development of therapeutic antitoxins and pharmacological
antidotes, and in some aspects of diagnostic test development. Such
assays are generally limited by several features. First, many cell
lines are insensitive to a toxin, probably because they lack one or
more surface receptors necessary for the toxin to enter the cell.
Second, even when immortalized cell lines, such as neuroblastoma
lines, are susceptible to intoxication, they are generally much
less sensitive than primary neuronal cells. Thirdly, most cell
lines that are sensitive to intoxication by one toxin are
insensitive to most other toxins, limiting their broad utility.
Finally, the use of holotoxin in the assays, that requires
substantial safety and regulatory issues, severely complicate their
use in high-throughput screening assays, e.g., it is difficult to
get enough toxin to perform the assays.
[0005] Hence, a need exists for efficient ways to intoxicate one or
more cells without having to use high concentrations of a
holotoxin. A further need exists to intoxicate cells that are
traditionally considered to be insensitive or refractory to toxins,
and to intoxicate more than one cell type with the same toxin. Yet
a further need exists to be able to intoxicate cells without using
the entire toxin, but rather the enzymatically active portion
thereof to avoid regulatory, safety and waste disposal issues.
SUMMARY OF THE INVENTION
[0006] The methods of the present invention relate to intoxicating
a cell in vitro with a toxin or an enzymatically active domain or
fragment thereof. As used herein, the term "toxin" refers to the
holotoxin, enzymatically active domains of a toxin, an
enzymatically active fragment of a toxin, and recombinant forms
thereof. The steps include preparing a mixture of a (e.g., one or
more) toxin or toxin fragment, and lipid or polymeric carrier, then
exposing the cell (e.g., one or more cells or cell types) with the
mixture to cause cell intoxication. In an embodiment, the toxin
includes or can be derived from (e.g., having mutations, deletions,
substitutions, truncations, and the like) any one or more of the
following toxins: botulinum neurotoxin, tetanospasmin,
tetrodotoxin, Clostridium difficile toxin Tcd A, Tcd B, Clostridium
Lethal Toxin, Anthrax Lethal Factor and edema factor, Ricin,
Exotoxin A, Diphtheria, Cholera, Tetanus toxins, Shiga toxin,
latrotoxin and a combination thereof. Enzymatically active portions
of these toxins can be used, as further described herein, and
include, e.g., light chain or chain A of a toxin. The light chain
or chain A, in an aspect, can have one or more mutations or
deletions. In an embodiment, the botulinum neurotoxin (e.g.,
serotypes A-G including any isoforms) or light chain portion is
used with the methods of the present invention. The lipid or
polymeric carrier, in one aspect, includes one or more DNA
transfection reagents (e.g., polyethylenimine (PEI), FuGene,
Lipofectamine, or any combination thereof). In one embodiment, the
cells are subjected to or come into contact with about 0.1 pM and
about 1 .mu.M of toxin or toxin fragment (e.g., about 1 nM and
about 10 nM) for a length of time between about 5 minutes and about
72 hours (e.g., about 1 hour and about 6 hours).
[0007] The methods of the present invention relate to delivering a
botulinum neurotoxin (e.g., one or more of the serotypes A-G and/or
isoforms thereof) or an enzymatically active fragment thereof to
the inside of a cell in vitro. The methods encompass contacting the
toxin or enzymatically active fragment (e.g., toxin light chain
protease) with a lipid or polymeric carrier prior to contacting the
cell with the toxin or toxin fragment. These steps result in the
cell being intoxicated with the botulinum neurotoxin or
enzymatically active fragment thereof (e.g., light chain portion
thereof). Steps of an embodiment of the invention further include
assessing the level of cleavage of one or more of the following to
determine the level of cell intoxication: a SNARE protein,
synaptobrevin 2, syntaxin and SNAP 25.
[0008] Yet another aspect of the present invention includes methods
of assessing an effect of a molecule, compound, drug, or condition,
in vitro, on a cell intoxicated with a toxin or fragment thereof.
The methods involve intoxicating one or more cells, as described
herein, and subjecting the intoxicated cells to the molecule,
compound, drug, or condition to be assessed, and assessing the
effect on the intoxicated cells of said molecule, compound, drug,
or condition. The test agent might be added before, during or after
cell intoxication. Assessing the effect of the molecule, compound,
drug or condition can be done using various methods of assessing
toxin effects including, e.g., Fluorescence Resonance Energy
Transfer (FRET) assays or release assays. The molecule, compound,
drug or condition can be an antagonist or an agonist of the toxin.
In one aspect, the effect is assessed by determining the percent
cleavage of the toxin substrate such as SNAP25, or assessing the
level of one or more substrate proteins, such as synaptobrevin
(e.g., 1 and 2), and syntaxin 1a.
[0009] In the case of a FRET assay, the steps include labeling the
toxin substrate with a donor fluorophore, to thereby obtain a
treated toxin substrate, exciting the donor fluorophore; and
determining resonance energy transfer of the treated toxin
substrate to a control substrate, wherein a difference in resonance
energy transfer of the treated toxin substrate as compared to the
control substrate is indicative of toxin protease activity.
[0010] In the case of using a release assay, the methods of the
present invention include utilizing an insulinoma cell line, which
is subjected to the toxin and a lipid or polymeric carrier, as
described herein, to obtain a mixture. The mixture is subjected to
glucose under conditions that allow for insulin secretion to occur.
The release of the toxin substrate is assessed by assessing the
level of insulin secretion. An increase of insulin is indicative of
an increase in toxin release, and a decrease of insulin is
indicative of a decrease in toxin release.
[0011] The present invention further embodies systems or kits for
delivering a toxin or toxin fragment to the inside of a cell in
vitro. The systems or kits include one or more toxins, and one or
more lipid or polymeric carriers (e.g., DNA transfection reagents).
In an embodiment, the system or kit includes one or more cell
lines, e.g., intoxicated with the toxin.
[0012] There are several advantages of the invention. The present
invention dramatically increases the number of cell lines available
for toxin assays and substantially broadens the range of toxins
that can be assayed within these different cell lines. It will also
increase the sensitivity of these cell lines to a toxin or toxin
fragment which will improve their utility and ability to detect
lower doses of the toxin. Furthermore, it allows rapid testing of
enzymatically active mutants (LC active mutants of toxins) at
physiologically relevant levels in the context of the mammalian
cellular environment. More importantly, it should remove the need
to use holotoxin (e.g., the entire toxin) to achieve intoxication,
thereby eliminating the safety and regulatory issues that otherwise
substantially complicate holotoxin use in a high-throughput
screen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a photograph of a western blot showing
pre-incubation of BoNT/A with a DNA transfection reagent that
enhances BoNT/A intoxication of two neuroblastoma cell lines.
Neuroblastoma cells M17 or Neuro2a (N2a) were incubated with 10 nM
BoNT/A for 3 or 24 hrs with (+) or without (-) pre-incubation of
toxin with FuGene-HD transfection reagent. Cells were washed and
cultured a further 24 hours, then protein was extracted, resolved
by SDS-PAGE and a Western blot was performed to detect SNAP25. The
extent of BoNT intoxication is measured by the extent of SNAP25
cleavage by BoNT/A protease. The native 25 kDa SNAP25 and the 24 kD
cleavage product of BoNT/A are indicated by arrows.
[0014] FIG. 2 is a photograph of a western blot showing a DNA
transfection reagent that facilitates rapid BoNT/A internalization
by Neuro2a cells. Neuro2a cells were exposed to 10 nM BoNT/A toxin
for 0.5, 1, 2, 3, 6 or 24 hrs with (+) or without (-)
pre-incubation of toxin with FuGene-HD transfection reagent. After
the indicated toxin exposure time, cells were harvested and cell
extracts were prepared. Proteins were resolved by SDS-PAGE and
SNAP25 cleavage was detected by Western blotting.
[0015] FIG. 3 is a photograph of a western blot showing DNA
transfection reagent that mediated BoNT/A intoxication by Neuro2a
cells is toxin concentration dependent. Neuro2a cells were exposed
to 10 nM, 1 nM and 0.1 nM of BoNT/A toxin for 24 hrs with (+) or
without (-) pre-incubation with FuGene-HD transfection reagent.
Cell extracts were prepared and the extent of BoNT/A intoxication
was measured by Western blotting to monitor SNAP25 cleavage.
[0016] FIG. 4 is a photograph of a western blot showing DNA
transfection reagent that facilitates BoNT/A internalization of
non-neuronal cell lines. Neuronal cell lines Neuro2a (N2A), M17 and
non-neuronal cell lines HEK293 (293), HIT-T15 were exposed to 10 nM
BoNT/A for 24 hrs with (+) or without (-) pre-incubation with DNA
transfection reagents, FuGene-HD or Lipofectamine 2000. Cell
extracts were prepared and the extent of BoNT/A intoxication was
measured by Western blotting to monitor SNAP25 cleavage.
[0017] FIG. 5 is a photograph of a western blot of various DNA
transfection reagents that promote cellular internalization of
BoNT/A Lc protease in the absence of Hc. Neuronal cell lines
Neuro2a (N2A), M17 and non-neuronal cell lines HIT-T15, HEK293
(293) were exposed for 24 hrs to 30 nM of recombinant BoNT/A LC
protease, using either the full-length protease (Lc438) or the
carboxyl-truncated form (Lc424). The protease was added to cell
culture with (+) or without (-) pre-incubation with the DNA
transfection reagents, FuGene-HD or Lipofectamine 2000. Cell
extracts were prepared and the extent of BoNT/A intoxication was
measured by Western blotting to monitor SNAP25 cleavage. Cell
extracts were prepared and the extent of BoNT/A intoxication was
measured by Western blotting to monitor SNAP25 cleavage.
[0018] FIG. 6 is a photograph of a western blot of defined cationic
lipid polymer reagents that promote BoNT intoxication. Neuronal
cell lines Neuro2a and M17 were incubated for 24 hrs with 30 nM
recombinant BoNT/A LC (Lc438) and a GFP expression plasmid with (+)
or without (-) pre-incubation with FuGene-HD (FuGene) or PEI having
average molecular weights of 10000, 25000 or 70000. Cell extracts
were prepared and the extent of BoNT/A intoxication was measured by
Western blotting to monitor SNAP25 cleavage. The efficiency of DNA
transfection was assayed by Western blotting.
[0019] FIG. 7 is a photograph of a western blot showing that
Bafilomycin inhibits DNA transfection reagent-mediated enhancement
of BoNT/A holotoxin and Lc internalization into cells. Primary
cells RCGN, neuronal cell lines Neuro2a, M17 and non-neuronal cell
lines 293HEK, HIT-T15 were treated with Bafilomycin for 2 hrs and
washed with DPBS before exposed to 10 nM of BoNT/A toxin for 4 or
24 hrs or 30 nM of Lc438 for 24 hrs + or - the FuGene transfection
reagent. Control cells were incubated with 10 nM of BoNT/A toxin or
30 nM of Lc438 + or - the FuGene transfection reagent without
pre-exposure cells with Bafilomycin. Cell extracts were prepared
after BoNT/A or Lc438 exposure and subjected to SDS-PAGE.
Bafilomycin effect on BoNT/A or Lc438 internalization was detected
by comparing the presence of 24 KDa SNAP25 by Western blotting.
[0020] FIG. 8 is a photograph of a western blot showing that DNA
transfection reagent enhances multiple toxin/Lc serotypes
internalization in two neuroblastoma cell lines. M17 or Neuro2a
cells were exposed to 50, 10 or 2 nM of BoNT/E toxin (A) or 30 or 6
nM of GST-Lc/E (B) or 50 or 10 nM of BoNT/B toxin (C) or 150 or 30
nM of recombinant Lc/B (D)+ or - the FuGene-HD transfection reagent
for 24 h. Cell extracts were prepared and revolved by SDS-PAGE.
Toxin or Lc internalization was measured by detecting the 24 KDa
SNAP25 (type E) or the reduction of VAMP2 (type B) by Western
blotting.
[0021] FIGS. 9A-K is a schematic of nucleic acid and amino acid
sequences of BoNT/A1 LC (SEQ ID NO: 1 and 2), BoNT/A2 LC (SEQ ID
NO: 3 and 4), BoNT/A3 LC (SEQ ID NO: 5 and 6), Botulinum B1 Okra
P10844 (SEQ ID NO: 7 and 8), Botulinum B2 Eklund 17B (SEQ ID NO: 9
and 10), Neurotoxin type C1 Clostridium botulinum (BoNT/C1) LC (SEQ
ID NO: 11 and 12), Neurotoxin type D Clostridium botulinum (BoNT/D)
LC (SEQ ID NO: 13 and 14), Neurotoxin type E Clostridium botulinum
(BoNT/E) LC (SEQ ID NO: 15 and 16), Neurotoxin type F Clostridium
botulinum (BoNT/F1) LC (SEQ ID NO: 17 and 18), Neurotoxin type F
Clostridium baratii (BoNT/F2) (SEQ ID NO: 19 and 20), Neurotoxin
type G Clostridium botulinum (BoNT/G) (SEQ ID NO: 21 and 22).
DETAILED DESCRIPTION OF THE INVENTION
[0022] A description of preferred embodiments of the invention
follows.
[0023] The present invention relates to methods for intoxicating a
cell with a toxin, or an enzymatically active fragment thereof. The
methods of the present invention allow one to easily and to a
greater extent intoxicate cells with toxin or toxin fragment. In an
embodiment, the methods of the present invention allow for the
intoxication of cells with only enzymatically active fragments of
the toxin, rather than the entire toxin. Utilizing only the
functional or enzymatically active portion of the toxin rather than
the entire toxin is advantageous because the enzymatically active
portion is easier to obtain and safer to work with. Furthermore,
the methods and systems of the present invention allow one to
intoxicate toxin insensitive cells, cells that could not otherwise
be intoxicated.
[0024] Accordingly, the methods of the present invention involve
intoxicating a cell with a toxin, the enzymatically active fragment
of the toxin, or a recombinant SNARE endoprotease. Intoxicating a
cell refers to getting the toxin or an enzymatically active
fragment thereof inside the cell. In particular, intoxicating a
cell refers to bringing the toxin or an enzymatically active
portion thereof past the cellular membrane and to its site of
action within the cell. In an embodiment, the toxin is carried from
the cell membrane into the cell with an endosome via endocytosis.
With respect to the present invention, endocytosis is a process
where cells engulf the toxin or fragment thereof at the cell
membrane. Once inside the cell, the endosome releases the toxin
enzyme active domain into the cytosol, the internal fluid of the
cell. After release, the toxin enzyme active domain is free to
cleave SNARE proteins, or undergo other cleavages or mechanisms of
action. As further described herein, the cell based assays measure
the release of the toxin enzyme active domain into the cytosol, as
well as measure cleavage of various proteins. These measurements
are performed to determine the toxin or its fragment's ability to
intoxicate the cell, and/or assess the effect of one or more
compounds, molecules, drugs, or conditions on the toxin or fragment
thereof.
Toxin, Enzymatically Active Portion Thereof and the Recombinant
Snare Endoprotease
[0025] The toxin that is used in the present invention refers to
the native toxin or the enzymatically active portion of the toxin.
The toxin used for preparing cells for a cell-based assay can be
any toxin known or later discovered or developed. A toxin is a
molecule, generally produced by a living cell or organism, that
gets into the cell and causes disease or injury. Certain toxins
come from animals such as spiders, snakes, pufferfish, scorpions,
jellyfish, and bees. Types of toxins include, e.g., neurotoxins
(e.g., Botulinum neurotoxin), and other toxins which effect other
cell types in addition to neurons such as cholera toxin,
clostridial difficile toxin (Tcd), anthrax lethal factor and edema
factor. A neurotoxin is a toxin that affects neurons. Several
toxins generally interact with membrane proteins such as ion
channels (e.g., sodium, potassium, or calcium channels). A common
effect is paralysis, which often sets in very rapidly. Examples of
neurotoxins include botulinum neurotoxin (BoNT), tetanospasmin,
tetrodotoxin. Other toxins implicated by the invention are,
Clostridium difficile toxin Tcd A, Tcd B, Clostridium Lethal Toxin,
Anthrax Lethal Factor and edema factor, Ricin, Exotoxin A,
Diphtheria, Cholera, Tetanus toxins, Shiga toxin, latrotoxin and a
combination thereof.
[0026] In an embodiment, BoNT or an enzymatically active portion
thereof is used. BoNT is a neurotoxin protein produced by the
bacterium Clostridium botulinum. There are at least seven different
BoNT serotypes (A to G), and some of the serotypes have various
isotypes (e.g., three isotypes of serotype A have been described).
Generally, the BoNT has two chains, a heavy chain (e.g., about
100-kDa) and a light chain (e.g., about 50-kDa) joined by a
disulfide bond. The heavy chain is a cell binding/translocation
domain that allows for the toxin to bind to and enter the cell.
[0027] The light chain is an enzyme (e.g., a protease) that cleaves
a fusion protein (e.g., SNAP-25, syntaxin or synaptobrevin) at a
neuromuscular junction, preventing vesicles from anchoring to the
membrane to release acetylcholine. By inhibiting acetylcholine
release, the toxin interferes with nerve impulses and causes
paralysis of muscles, seen in botulism.
[0028] The term "toxin" or "fragment" for use with the present
invention includes derivatives of a toxin's enzymatically active
portion. A "derivative" refers to a molecule with toxin enzymatic
activity but contains one or more chemical or functional
alterations thereof, as compared to the native enzymatic portion.
For instance, the botulinum toxin light chain protease can be
modified so that one or more of its amino acid residues is deleted,
modified, replaced, or truncated. For instance, the botulinum toxin
light chain protease can be modified in a way such that, for
instance, the modification enhances its properties or decreases
undesirable side effects, but that still retains the desired
botulinum toxin activity. The botulinum toxin can be derived from
any of the botulinum toxin serotypes and/or isoforms produced by
the bacterium. Alternatively, the botulinum toxin can be a toxin
prepared using recombinant or synthetic chemical techniques (e.g.,
a recombinant peptide, a fusion protein, or a hybrid neurotoxin, as
prepared from subunits or domains of different botulinum toxin
serotypes). Additionally, the botulinum toxin can be in the form of
a botulinum toxin precursor, which can itself be non-toxic, for
instance a non-toxic zinc protease that becomes toxic on
proteolytic cleavage.
[0029] "Enzymatically active" portion or fragment of the toxin
refers to the portion or domain of the toxin that normally gets
into the inside of the cell (e.g., in the endosome or cytosol) and
is active. Toxins are often made up of at least two parts, a
cell-binding/translocation domain, and an enzymatically active
domain. In the BoNT, the enzymatically active domain is often
referred to as the "light chain." However, the enzymatically active
domain for other toxins can have other names. For example, with the
ricin toxin, the enzymatically active domain is the "A" Chain. The
cell-binding/translocation domain facilitates binding of the toxin
to the cell membrane and transporting the toxin across the cellular
membrane. For certain toxins like the BoNT, this domain is referred
to as the heavy chain. For other toxins, such as ricin, this is
referred to as the B Chain.
[0030] In an embodiment, enzymatically active refers to a protein
that causes the cleavage of one or more proteins in the cell, which
in turn causes toxic effects. In the case of certain toxins, the
enzymatically active domain cleaves a SNARE ("Soluble NSF
Attachment REceptor") protein. SNARE proteins are a large protein
superfamily consisting of several members. The primary role of
SNARE proteins is to mediate fusion of cellular transport vesicles
with the cell membrane. The core SNARE complex is formed by four
.alpha.-helices contributed by synaptobrevin, syntaxin and SNAP-25.
Different toxins, serotypes of a certain toxin, or cell types will
involve cleavage of different SNARE proteins. Tetanospasmin, e.g.,
is the neurotoxin produced by the vegetative spore of Clostridium
tetani and causes tetanus. BoNT A, C, and E cleave SNAP-25, in
addition BoNT C cleaves syntaxin 1. BoNT B, D, F, G and tetanus
toxin cleave VAMP 1 and 2 isoforms.
[0031] Botulinum toxin is generally considered to be a
zinc-dependent protease. As described herein, enzymatic activity
resides generally in the light chain of the molecules. These
enzymes cleave SNARE proteins, synaptobrevin 1 and 2, syntaxin and
SNAP 25, which form the core of a complex involved in the fusion of
transmitter-containing vesicles with the plasma membrane. Prior to
fusion, the SNARE proteins in the vesicle and plasma membrane
interact forming a complex which contracts with an increase in the
intracellular calcium concentration, pulling the vesicle close to
the plasma membrane. Interaction between lipids in the two
membranes allows the vesicle and nerve terminal active zone to
fuse. During this fusion, the contents of the vesicles, mainly
neurotransmitters, are released, and the inner surface of the
vesicles is exposed to the synaptic cleft. If one of the SNARE
proteins is cleaved by a neurotoxin, complex formation cannot occur
and fusion is interrupted.
[0032] The present invention further involves using a toxin
substrate of a recombinant SNARE protein. Any one of the SNARE
proteins can be made using DNA recombinant technology. With respect
to the BoNT serotypes, the light chain for each serotype has an
amino acid sequence, or is encoded by a nucleic acid sequence as
shown in FIGS. 9A-C. The present invention specifically relates to
intoxicating cells with the light chain of any of the BoNT
serotypes, as well as any recombinant, mutated, truncated or
deleted portions thereof. As such, the toxin or fragment thereof
can be the recombinant form of any toxin, the enzymatically active
portion thereof (e.g., the light chain of a BoNT serotype), or a
SNARE protein. The toxin substrate can be made from recombinant DNA
which transcribes the desired amino acid sequence of the toxin or
fragment thereof. The recombinant nucleic acid sequence can be a
nucleotide "variant" of any toxin or fragment thereof (e.g., toxin,
enzymatically active portion thereof, or SNARE protein). A variant
is a sequence that differs from the known nucleotide sequence for
that molecule in having a truncation, and/or one or more nucleotide
deletions, substitutions or additions. Such modifications can be
readily introduced using standard mutagenesis techniques, such as
oligonucleotide-directed site-specific mutagenesis as taught, for
example, by Adelman et al. (DNA 2:183, 1983). Nucleotide variants
can be naturally occurring allelic variants, or non-naturally
occurring variants. Variant nucleotide sequences preferably exhibit
at least about 70%, more preferably at least about 80% and most
preferably at least about 90% homology to the recited sequence.
Such variant nucleotide sequences will generally hybridize to the
recited nucleotide sequence under stringent conditions. In one
embodiment, "stringent conditions" refers to prewashing in a
solution of 6.times.SSC, 0.2% SDS; hybridizing at 65.degree.
Celsius, 6.times.SSC, 0.2% SDS overnight; followed by two washes of
30 minutes each in 1.times.SSC, 0.1% SDS at 65.degree. C. and two
washes of 30 minutes each in 0.2.times.SSC, 0.1% SDS at 65.degree.
C.
Cells Intoxicated with the Toxin or Fragment Thereof
[0033] An aspect of the invention are the cells that are
intoxicated with the toxin or enzymatically active fragment
thereof. The toxin or toxin enzyme active fragment and the lipid or
polymeric carrier come into contact with one or more cells which
allows the toxin to enter the cells. When a cell is intoxicated
with the toxin, in an aspect, the cell is prepared for a cell based
assay, e.g., to evaluate a drug candidate. "Preparing" a cell
refers to intoxicating one or more cells or cell types with a toxin
or fragment so that the cell is ready for a cell based assay.
[0034] Various cell types can be used with the methods of the
present invention. In an embodiment, cell types include those that
are sensitive to intoxication by a toxin as well as those that are
insensitive. Any cell type known or later discovered or developed
can be used with the present invention so long as the cell comes
into contact with the toxin or fragment thereof. Cells that are
normally insensitive include all non-neuroendocrine immortalized
cell lines, many neuroblastoma, and neuronal cell lines and a
variety of stem cells.
[0035] Aspects of the present invention provide, in part, a cell
that can be intoxicated by a toxin using a lipid or polymeric
carrier. As used herein, the term "cell," means any eukaryotic cell
that can be intoxicated with a toxin using a lipid or polymeric
carrier, as described herein. The term cell encompasses cells from
a variety of organisms, such as, e.g., murine, rat, porcine,
bovine, equine, primate and human cells; from a variety of cell
types such as, e.g., neural and non-neural; and can be isolated
from or part of a heterogeneous cell population, tissue or
organism. Cells useful in aspects of the present invention can
include, e.g., primary cells; cultured cells; established cells;
normal cells; transformed cells; tumor cells; infected cells;
proliferating and terminally differentiated cells; and stably or
transiently transfected cells, including stably and transiently
transfected cells.
[0036] A cell that is intoxicated by a toxin or toxin enzyme active
fragment is a cell that has internalized the toxin enzyme active
domain in a manner such that the natural substrate of the enzyme
within the cell can be acted on by the enzyme. Cells utilized in
the methods of the present invention include any cell containing
the substrate of a toxin enzyme active domain, both neuronal and
non-neuronal cells.
[0037] Neuronal cells useful in aspects of the invention include,
without limitation, primary neuronal cells; immortalized or
established neuronal cells; transformed neuronal cells; neuronal
tumor cells; stably and transiently transfected neuronal cells and
further include, yet are not limited to, mammalian, murine, rat,
primate and human neuronal cells. Examples of neuronal cells useful
in aspects of the invention include, e.g., peripheral neuronal
cells, such as, e.g., motor neurons and sensory neurons; and CNS
neuronal cells, such as, e.g., spinal cord neurons like embryonic
spinal cord neurons, dorsal root ganglia (DRG) neurons, cerebral
cortex neurons, cerebellar neurons, hippocampal neurons and motor
neurons. Neuronal cells useful in the invention can be, for
example, central nervous system (CNS) neurons; neuroblastoma cells;
motor neurons, hippocampal neurons or cerebellar neurons and
further can be, without limitation, Neuro-2A, SH-SY5Y, NG108-15,
N1E-115 or SK-N-DZ cells. These and additional primary and
established neurons can be useful in carrying out the methods of
the present invention.
[0038] Neurons useful in aspects of the invention include, without
limitation, primary cultures such as primary cultures of embryonic
dorsal root ganglion (DRG) neurons. As one example, primary
cultures of embryonic rat DRG neurons are described in Mary J.
Welch et al., Sensitivity of embryonic rat dorsal root ganglia
neurons to Clostridium botulinum neurotoxins, 38(2) Toxicon 245 258
(2000); and primary cultures of fetal spinal cord neurons, for
example, primary cultures of murine fetal spinal cord neurons are
described in Elaine A. Neale et al., Botulinum neurotoxin A blocks
synaptic vesicle exocytosis but not endocytosis at the nerve
terminal, 147(6) J. Cell Biol. 1249-1260 (1999), and John A.
Chaddock et al., Inhibition of vesicular secretion in both neuronal
and non-neuronal cells by a retargeted endopeptidase derivative of
Clostridium botulinum neurotoxin type A, 68(5) Infect. Immun.
2587-2593 (2000).
[0039] Neuronal cell lines useful in carrying out the methods of
the present invention include, without limitation, neuroblastoma
cell lines, neuronal hybrid cell lines, spinal cord cell lines,
central nervous system cell lines, cerebral cortex cell lines,
dorsal root ganglion cell lines, hippocampal cell lines and
pheochromocytoma cell lines.
[0040] Neuroblastoma cell lines, such as, e.g., murine, rat,
primate or human neuroblastoma cell lines can be useful in aspects
of the invention. Neuroblastoma cell lines useful in aspects of the
invention include, without limitation, BE(2)-C (ATCC CRL-2268;
ECACC 95011817), BE(2)-M17 (ATCC CRL-2267; ECACC 95011816), C1300
(ECACC 93120817), CHP-212 (ATCC CRL-2273), CHP-126 (DSMZ ACC 304),
IMR 32 (ATCC CRL-127; ECACC 86041809; DSMZ ACC 165), KELLY (ECACC
92110411; DSMZ ACC 355), LA-N-2, see, e.g., Robert C. Seeger et
al., Morphology, growth, chromosomal pattern and fibrinolytic
activity of two new human neuroblastoma cell lines, 37(5) Cancer
Res. 1364-1371 (1977); and G. J. West et al., Adrenergic,
cholinergic, and inactive human neuroblastoma cell lines with the
action-potential Na+ ionophore, 37(5) Cancer Res. 1372-1376 (1977),
MC-IXC (ATCC CRL-2270), MHH-NB-11 (DSMZ ACC 157), N18Tg2 (DSMZ ACC
103), N1E-115 (ATCC CCL-2263; ECACC 88112303), N4TG3 (DSMZ ACC
101), Neuro-2A (ATCC CCL-131; ECACC 89121404; DSMZ ACC 148), NB41A3
(ATCC CCL-147; ECACC 89121405), NS20Y (DSMZ ACC 94), SH-SY5Y (ATCC
CRL-2266; ECACC 94030304; DSMZ ACC 209), SIMA (DSMZ ACC 164),
SK-N-DZ (ATCC CRL-2149; ECACC 94092305), SK-N-F1 (ATCC CRL-2142,
ECACC 94092304), SK-N-MC (ATCC HTB-10, DSMZ ACC 203) and SK-N-SH
(ATCC HTB-11, ECACC 86012802).
[0041] Neuronal hybrid cell lines, such as, e.g., murine, rat,
primate and human hybrid neuronal cell lines can be useful in
aspects of the invention. Such hybrid cell lines include
neuroblastoma/glioma hybrids, such as, e.g., N18 (ECACC 88112301),
NG108-15 (ATCC HB-12317, ECACC 88112302) and NG115-401L (ECACC
87032003); neuroblastoma/motor neuron hybrids, such as, e.g.,
NSC-19 and NSC-34, which express motor neuron characteristics,
display a multipolar neuron-like phenotype, express high levels of
choline acetyltransferase (CHAT), generate action potentials,
express neurofilament triplet proteins and synthesize, store and
release acetylcholine, see, e.g., N. R. Cashman et al.,
Neuroblastoma.times.spinal cord (NSC) hybrid cell lines resemble
developing motor neurons, 194(3) Dev. Dyn. 209-221 (1992); and
Christopher J. Eggett et al., Development and characterisation of a
glutamate-sensitive motor neuronal cell line, 74(5) J. Neurochem.
1895-1902 (2000); neuroblastoma/root ganglion neuron hybrids, such
as, e.g., F11, see, e.g., Doros Platika et al., Neuronal traits of
clonal cell lines derived by fusion of dorsal root ganglia neurons
with neuroblastoma cells, 82(10) Proc. Natl. Acad. Sci. U.S.A.
3499-3503 (1985), ND-E (ECACC 92090915), ND-ill (ECACC 92090916),
ND7/23 (ECACC 92090903), ND8/34 (ECACC 92090904) and ND27 (ECACC
92090912); neuroblastoma/hippocampal neuron hybrids, such as, e.g.,
HN-33, see, e.g., Henry J. Lee et al., Neuronal properties and
trophic activities of immortalized hippocampal cells from embryonic
and young adult mice. 10(6) J. Neurosci. 1779-1787 (1990). In
further aspects of this embodiment, a neuroblastoma/motor neuron
hybrid can be, e.g., NSC-19 and NSC-32. In further aspects of this
embodiment, a neuroblastoma/root ganglion neuron hybrid can be,
e.g., F11, ND-E, ND-U1, ND7/23, ND8/34 and ND27. In further aspects
of this embodiment, a neuroblastoma/hippocampal neuron hybrid can
be, e.g., HN-33.
[0042] Spinal cord cell lines, such as, e.g., murine, rat, primate
or human spinal cord cell lines can be useful in aspects of the
invention and include, without limitation, TE 189.T (ATCC CRL-7947)
and M4b, see, e.g., Ana M. Cardenas et al., Establishment and
characterization of immortalized neuronal cell lines derived from
the spinal cord of normal and trisomy 16 fetal mice, an animal
model of Down syndrome, 68(1) J. Neurosci. Res. 46-58 (2002). As an
example, a human spinal cord cell line can be generated from
precursors of human embryonic spinal cord cells (first trimester
embryos) that are immortalized with a tetracycline repressible
v-myc oncogene as described in Ronghao Li et al., Motoneuron
differentiation of immortalized human spinal cord cell lines, 59(3)
J. Neurosci. Res. 342-352 (2000). Such cells can be expanded
indefinitely in proliferative growth conditions before rapid
differentiation (4-7 days) into functional neurons that express
neuronal phenotypic markers such as choline acetyltransferase. As
another example, a murine spinal cord cell line can be prepared by
immortalizing an embryonic spinal cord culture using transforming
media. Such a spinal cord cell line can be, for example, the murine
M4b line and can express neuronal markers such as NSE,
synaptophysin, MAP 2 and choline acetyltransferase, and can release
acetylcholine upon appropriate stimulation, see, e.g., Cardenas et
al., supra, (2002).
[0043] Central nervous system (CNS) cell lines, such as, e.g.,
murine, rat, primate and human CNS cell lines, can be useful in
aspects of the invention. A useful CNS cell line can be, for
example, a human CNS cell line immortalized with a tetracycline
repressible v-myc oncogene as described in Dinah W. Sah et al.,
Bipotent progenitor cell lines from the human CNS, 15(6) Nat.
Biotechnol. 574-580 (1997). Upon repression of the oncogene, the
cells differentiate into neurons.
[0044] Cerebral cortex cell lines, such as, e.g., murine, rat,
primate and human cerebral cortex cell lines, can be useful in
aspects of the invention and include, without limitation, CNh, see,
e.g., Ana M. Cardenas et al., Calcium signals in cell lines derived
from the cerebral cortex of normal and trisomy 16 mice, 10(2)
Neuroreport 363-369 (1999), HCN-1a (ATCC CRL-10442) and HCN-2 (ATCC
CRL-10742). As an example, murine cortex primary cultures from
12-16 days embryos can be immortalized by culturing the cells in
conditioned media from a rat thyroid cell line that induces
transformation in vitro. The immortalized cells can be
differentiated into neurons expressing neuronal markers using the
appropriate media; these differentiated cells express choline
acetyltransferase and secrete acetylcholine and glutamate in
response to depolarization and nicotine stimulation, see, e.g.,
David D. Allen et al., Impaired cholinergic function in cell lines
derived from the cerebral cortex of normal and trisomy 16 mice,
12(9) Eur. J. Neurosci. 3259-3264 (2000).
[0045] Dorsal root ganglia cell lines, such as, e.g., murine, rat,
primate and human dorsal root ganglia cell lines, can be useful in
aspects of the invention and include, without limitation, G4b, see,
e.g., David D. Allen et al., A dorsal root ganglia cell line
derived from trisomy 16 fetal mice, a model for Down syndrome,
13(4) Neuroreport 491-496 (2002). Embryonic dorsal root ganglia
primary cultures can be immortalized with transforming conditioned
media as described above. Upon differentiation, the cell line
exhibits neuronal traits and lacks glial markers by
immunohistochemistry. Release of neurotransmitters such as
acetylcholine can be induced in response to potassium and nicotine,
see, e.g., Allen et al., supra, (2002).
[0046] Hippocampal cell lines, such as, e.g., murine, rat, primate
and human hippocampal lines can be useful in aspects of the
invention and include, without limitation, HT-4, see, e.g., K.
Frederiksen et al., Immortalization of precursor cells from the
mammalian CNS, 1(6) Neuron 439-448 (1988) and HT-22, see, e.g.,
John B. Davis and Pamela Maher, Protein kinase C activation
inhibits glutamate-induced cytotoxicity in a neuronal cell line,
652(1) Brain Res. 169-173 (1994). As an example, the murine
hippocampal cell line HT-22 can be useful in the invention. As a
further non-limiting example, the immortalized HN33 hippocampal
cell line can be useful in the invention. This hippocampal cell
line was derived from the fusion of primary neurons from the
hippocampus of postnatal day 21 mice with the N18TG2 neuroblastoma
cell line, and, when differentiated, shares membrane properties
with adult hippocampal neurons in primary culture, see, e.g., Henry
J. Lee et al., Neuronal Properties and Trophic Activities of
Immortalized Hippocampal Cells from Embryonic and Young Adult Mice,
19(6) J. Neurosci. 1779-1787 (1990); and Henry J. Lee et al.,
Immortalized young adult neurons from the septal region: generation
and characterization, 52(1-2) Brain Res. Dev Brain Res. 219-228
(1990).
[0047] A variety of non-neuronal cells are used to carry out the
steps of the present invention. Non-neuronal cells useful in
aspects of the invention include, e.g., primary non-neuronal cells;
immortalized or established non-neuronal cells; transformed
non-neuronal cells; non-neuronal tumor cells; stably and
transiently transfected non-neuronal cells and further include, yet
are not limited to, mammalian, murine, rat, primate and human
non-neuronal cells. Non-neuronal cells useful in aspects of the
invention further include, for example, any of the following
primary or established cells: anterior pituitary cells; adrenal
cells, such as, e.g., chromaffin cells of the adrenal medulla;
pancreatic cells, such as. e.g., pancreatic acinar cells,
pancreatic islet cells and insulinoma HIT or INS-1 cells; ovarian
cells, such as, e.g., steroid-producing ovarian cells; kidney
cells, such as. e.g., inner medullary collecting duct (IMCD) cells;
stomach cells, such as, e.g., enterochromaffin cells; blood cells,
such as. e.g., eurythrocytes, leucocytes, platelets, neutrophils,
eosinophils, mast cells; epithelial cells, such as. e.g., those of
the apical plasma membrane; fibroblasts; thyroid cells;
chondrocytes; muscle cells; hepatocytes; glandular cells such as,
e.g., pituitary cells, adrenal cells, chromaffin cells; and cells
involved in glucose transporter (GLUT4) translocation. See e.g., US
Publication No.: 20080003240.
[0048] Accordingly, an aspect of the present invention relates to
intoxicating cells that are insensitive to or refractory to the
toxin or fragment thereof without the presence of a lipid or
polymeric carrier. Insensitive cells are cells that do not become
intoxicated or become intoxicated at low levels, with holotoxin or
fragment thereof in conditions that occur generally in nature, or
absent a lipid or polymeric carrier. Refractory cells, as used
herein, are cells that do not become intoxicated or resist
intoxication under the same conditions.
Lipid or Polymeric Carrier
[0049] The present invention utilizes one or more lipid carriers,
polymeric carriers, or a combination thereof to intoxicate the cell
with the toxin or fragment thereof. A "lipid carrier" or "polymeric
carrier" refers to a carrier that allows the toxin or fragment
thereof to pass through the cellular membrane. Lipofection is
generally used to inject genetic material into a cell by means of
liposomes have a phospholipid bilayer which merge with the cell
membrane. However, the methods of present invention unexpectedly
allow for the delivery of a toxin polypeptide, rather than nucleic
acid molecules. The carrier of the present invention, in an aspect,
can be cationic, anionic or neutrally charged. Examples of lipid
carriers include Lipofectamine (Invitrogen Corporation, Carlsbad,
Calif.), cardiolipins, or other cationic, anionic or neutrally
charged polymers.
[0050] A polymeric carrier is a carrier that has a repeating
backbone, which can be linear or branched. In an embodiment, the
linear backbone can have a primarily hydrocarbon backbone,
interspersed by heteroatoms such nitrogen, oxygen, sulfur, silicon
and phosphorus. Additionally, the backbone can also be a polymer of
other repeating units such as amino acids, poly(ethyleneoxy),
poly(propyleneamine), polyalkyleneimine, and a combination thereof.
The polymeric carrier can be configured to include chemical groups
to be positively charged, negatively charged, or have no overall
charge. In another embodiment, the backbone has attached a
plurality of side-chain moieties (e.g., ammonium groups, pyridinium
groups, phosphonium groups, sulfonium groups, guanidinium groups,
or amidinium groups) or are otherwise branched. See WO/2006/094263
for positively charged carriers.
[0051] In another embodiment, the carrier is a polylysine with
positively charged branching groups attached to the lysine
side-chain amino groups. The polylysine is commercially available
e.g., from Sigma Chemical Company, St. Louis, Mo. In another
embodiment, the carrier can have a polyethyleneimine (PEI)
backbone. For example; polylysine or polyethyleneimine (PEI)
backbone, which may be linear or branched, can be used and have a
molecular weight ranging from 0.5 kD to 250 kD. Other polycationic
polymer carriers could also be used. Another example of a polymeric
carrier includes Fugene (Hoffmann LaRoche, Ltd.).
[0052] DNA uptake facilitating agent is a reagent that allows
nucleic acid to be transfected into a cell. It is generally used to
get recombinant DNA into the cell. The present invention
surprisingly found the DNA uptake facilitating agent assists in
getting a totally different type of molecule, a toxin or fragment
thereof, into the cell. See Exemplification. Any DNA uptake
facilitating agent can be used, including those known in the art
and those later developed or discovered. Many DNA uptake
facilitating agents are carriers that cause DNA to get into a cell
e.g., via endocytosis, and, in an embodiment, are generally lipid
carriers or polymeric carriers. Examples of DNA uptake facilitating
agents includes transfection reagents such as Lipofectamine,
Dojindo Hilymax, Fugene, jetPEI, Effectene or DreamFect. As such,
the present invention involves contacting a cell/toxin mixture with
a DNA uptake facilitating agent, a lipid carrier, a polymeric
carrier and/or any combination thereof.
[0053] The present invention involves subjecting the toxin to
contact with the lipid or polymeric carrier prior to exposure to
the cell or cells. "Subjecting" or "exposing" the mixture to the
carrier refers to preparing the mixture for a sufficient time and
in an amount that allows for the intoxication of the cell with the
toxin or fragment thereof.
[0054] In an embodiment, the amount of toxin substrate will depend
in part on the type of cell based assay is being performed, the
compound being assayed. The amount of the toxin or fragment thereof
used, in an embodiment, ranges between about 0.1 pM and about 1
.mu.M, and preferably between about 1 nM and about 10 nM.
[0055] The amount of the carrier used depends on various factors
such as the amount of the length of time the mixture will be
exposed to the carrier, the potency or effectiveness of the
carrier, the recommended dosage of DNA transfection for use in
nucleic acid transfection, the type of medium, amount of toxin or
fragment thereof, and temperature. The amount of the carrier used,
in an embodiment, ranges between about 0.1 pM and about 1 .mu.M,
and preferably between about 1 nM and about 10 nM.
[0056] The amount of time the mixture is subjected to the carrier
for the present invention relates to the amount of time sufficient
to allow the cell to be intoxicated with the toxin substrate. In an
embodiment, the amount of time for subjecting the carrier to the
mixture ranges from about 5 minutes and about 72 hours, and
preferably from about 1 hour and about 6 hours.
Cell Based Assays:
[0057] The methods of the present invention can be used with any
type of cell based assay where toxin intoxication of a cell is
desirable. Cell based assays can be used to assess the effect of a
molecule, drug, compound or condition on a cell intoxicated with
the toxin or fragment thereof. Varying concentrations of molecules
or drugs can be used to determine their effect on a cell
intoxicated with the toxin. The molecule or drug being assessed
includes those that are antagonists and agonists. An antagonist is
a molecule that inhibits the toxin enzymatic activity (e.g.,
cleavage of a SNARE protein) or prevents release of the toxin
substrate from the endosome to its site of action. An agonist is a
molecule that increases these effects. In an embodiment, cell based
assays are those that can measure the extent of intoxication of the
toxin, its ability to enzymatically act on its substrate, and/or
extent of release of the toxin substrate by the endosome.
[0058] Aspects of the present invention provide, in part, detecting
the presence of enzymatic activity of contacted cell relative to a
control cell, where a difference in the activity of the contacted
cell as compared to the control cell is indicative of enzymatic
activity. As used herein, the term "control cell" means a cell of
the same or similar type as the contacted cell and grown under the
same conditions but which is not contacted with any sample or is
contacted with a defined negative sample or a defined positive
sample. A variety of control cells are useful in the methods
described herein and a control cell can be a positive control cell
or a negative control cell. A control cell can be, for example, a
negative control cell e.g., that lacks the toxin or toxin fragment.
A control cell also can be, for example, a positive control cell
that is fully intoxicated with the toxin.
[0059] A wide variety of assays can be used to determine the
presence of toxin activity, including direct and indirect assays
for toxin uptake. Assays that determine toxin binding or uptake
properties can be used to assess activity. Such assays include,
e.g., cross-linking assays using labeled toxin. Other assays
include immunocytochemical assays that detect toxin binding using
labeled or unlabeled antibodies, see, e.g., Atsushi Nishikawa et
al., The receptor and transporter for internalization of
Clostridium botulinum type C progenitor toxin into HT-29 cells,
319(2) Biochem. Biophys. Res. Commun. 327-333 (2004) and
immunoprecipitation assays, see, e.g., Yukako Fujinaga et al.,
Molecular characterization of binding subcomponents of Clostridium
botulinum type C progenitor toxin for intestinal epithelial cells
and erythrocytes, 150(Pt 5) Microbiology 1529-1538 (2004).
Antibodies useful for these assays include antibodies can be made
for the toxin enzyme domain modified substrate, such as a cleaved
SNARE protein, and its existence and level can be determined. If
the antibody is labeled, the binding of the molecule can be
detected by various means, including Western blotting, direct
microscopic observation of the cellular location of the antibody,
measurement of cell or substrate-bound antibody following a wash
step, or electrophoresis, employing techniques known to those of
skill in the art. If the antibody is unlabeled, one can employ a
labeled secondary antibody for indirect detection of the bound
molecule, and detection can proceed as for a labeled antibody.
These and similar assays that determine cleavage of a SNARE protein
or other proteins normally cleaved by the toxin can be used to
determine intoxication of the toxin.
[0060] Assays that monitor the release of a molecule after exposure
to toxin or toxin thereof can also be used to assess for the
presence of toxin activity. For example, an insulin release assay
disclosed herein can monitor the release of a molecule after
exposure to some toxins, and thereby be useful in assessing whether
intoxication has occurred. Other assays include methods that
measure inhibition of radio-labeled catecholamine release from
neurons, such as, e.g., .sup.3H noradrenaline or .sup.3H dopamine
release, see e.g., A Fassio et al., Evidence for calcium-dependent
vesicular transmitter release insensitive to tetanus toxin and
botulinum toxin type F, 90(3) Neuroscience 893-902 (1999); and Sara
Stigliani et al., The sensitivity of catecholamine release to
botulinum toxin C1 and E suggests selective targeting of vesicles
set into the readily releasable pool, 85(2) J. Neurochem. 409-421
(2003), or measures catecholamine release using a fluorometric
procedure, see, e.g., Anton de Paiva et al., A role for the
interchain disulfide or its participating thiols in the
internalization of botulinum neurotoxin A revealed by a toxin
derivative that binds to ecto-acceptors and inhibits transmitter
release intracellularly, 268(28) J. Biol. Chem. 20838-20844 (1993);
Gary W. Lawrence et al., Distinct exocytotic responses of intact
and permeabilised chromaffin cells after cleavage of the 25-kDa
synaptosomal-associated protein (SNAP-25) or synaptobrevin by
botulinum toxin A or B, 236(3) Eur. J. Biochem. 877-886 (1996); and
Patrick Foran et al., Botulinum neurotoxin C1 cleaves both syntaxin
and SNAP-25 in intact and permeabilized chromaffin cells:
correlation with its blockade of catecholamine release, 35(8)
Biochemistry 2630-2636 (1996); and methods that measure inhibition
of hormone release from endocrine cells, such as, e.g., anterior
pituitary cells or ovarian cells. Assays for determining toxin
substrate cleavage or assessing release that are known in the art
or later developed can be used with the methods of the present
invention.
[0061] In a particular embodiment, after intoxicating the cell with
the toxin or toxin fragment, an inhibition of insulin release assay
can be used to determine the presence of toxin activity in cells
that can secrete insulin; an inhibition of noradrenaline release
assay can be used to determine toxin activity in cells that secrete
noradrenaline; and an inhibition of estrogen release assay can be
used to determine toxin activity in cells that secrete
estrogen.
[0062] Assays that detect the cleavage of a toxin substrate can
also be used to assess for the presence of toxin activity. In these
assays, generation of a toxin cleavage-product is detected after
toxin treatment. As an example, a SNAP-25 cleavage assay can detect
the cleavage of a toxin substrate and thereby be useful in
assessing toxin activity (see Exemplification). Other methods
useful to detect the cleavage of a toxin substrate are described
in, e.g., Lance E. Steward et al., FRET Protease Assays for
Botulinum Serotype A/E Toxins, U.S. Patent Publication No.
2003/0143650 (Jul. 31, 2003); and Ester Fernandez-Salas et al.,
Cell-based Fluorescence Resonance Energy Transfer (FRET) Assays for
Clostridial Toxins, U.S. Patent Publication 2004/0072270 (Apr. 15,
2004). These and similar assays for toxin substrate cleavage can be
useful in assessing toxin activity.
[0063] Western blot analysis using an antibody that recognizes
toxin SNAP-25-cleaved product can be used to determine the presence
of toxin activity. Examples of anti-SNAP-25 antibodies useful for
these assays include, e.g., rabbit polyclonal anti-SNAP25.sub.197,
antiserum pAb anti-SNAP25.sub.197 #1 (Allergan, Inc., Irvine,
Calif.), mouse monoclonal anti-SNAP-25 antibody SMI-81 (Sternberger
Monoclonals, Lutherville, Md.), mouse monoclonal anti-SNAP-25
antibody CI 71.1 (Synaptic Systems, Goettingen, Germany), mouse
monoclonal anti-SNAP-25 antibody CI 71.2 (Synaptic Systems,
Goettingen, Germany), mouse monoclonal anti-SNAP-25 antibody SP12
(Abcam, Cambridge, Mass.), rabbit polyclonal anti-SNAP-25 antiserum
(Synaptic Systems, Goettingen, Germany), and rabbit polyclonal
anti-SNAP-25 antiserum (Abcam, Cambridge, Mass.).
[0064] Some toxins lead to cell death following intoxication and
the toxin enzyme active domain acting on its substrate. Cell death
might be the readout in such assays.
[0065] It is envisioned that a wide variety of processing formats
can be used in conjunction with the methods of the present
invention, including, for example, manual processing, partial
automated-processing, semi-automated-processing, full
automated-processing, high throughput processing, high content
processing, and any combination thereof. High throughput processing
is one preferred embodiment. See US Patent Publication No.
20080003240.
Fluorescence Resonance Energy Transfer (FRET)
[0066] The amount of intoxication by the toxin or fragment thereof
can be determined, in an embodiment, using Fluorescence Resonance
Energy Transfer (FRET). FRET is a distance-dependent interaction
between the electronic excited states of two molecules in which
excitation is transferred from a donor fluorophore to an acceptor
without emission of a photon. The process of energy transfer
results in a reduction (quenching) of fluorescence intensity and
excited state lifetime of the donor fluorophore and, where the
acceptor is a fluorophore, can produce an increase in the emission
intensity of the acceptor. Upon cleavage of the toxin substrate of
the invention, resonance energy transfer is reduced and can be
detected, for example, by increased donor fluorescence emission,
decreased acceptor fluorescence emission, or by a shift in the
emission maxima from near the acceptor emission maxima to near the
donor emission maxima. If desired, the amount of toxin substrate in
a sample can be calculated as a function of the difference in the
degree of FRET using the appropriate standards.
[0067] The toxin of the present invention can be formulated to
contain a donor fluorophore; an acceptor having an absorbance
spectrum overlapping the emission spectrum of the donor
fluorophore; and a toxin recognition sequence that includes a
cleavage site (e.g., a SNARE protein), wherein the cleavage site
intervenes between the donor fluorophore and the acceptor and
wherein, under the appropriate conditions, resonance energy
transfer is exhibited between the donor fluorophore and the
acceptor. Since, in an embodiment, the enzymatically active toxin
fragment or SNARE recombinant protein contains the cleavage
site.
[0068] A variety of donor fluorophores and acceptors, including
fluorescent and non-fluorescent acceptors, are useful preparing the
toxin substrates for carrying out the FRET assay. Donor
fluorophores useful in the invention include, but are not limited
to, fluorescein, ALEXA FLUOR.RTM. 488, DABCYL, and BODIPY.RTM..
Acceptors useful in the invention include, but are not limited to,
tetramethylrhodamine, EDANS and QSY.RTM.. Exemplary donor
fluorophoreacceptor pairs useful for inclusion in the toxin
substrate of the present invention include, without limitation,
fluorescein-tetramethylrhodamine, ALEXA FLUOR.RTM.
488-tetramethylrhodamine, DABCYL-EDANS, fluorescein-QSY.RTM. 7, and
ALEXA FLUOR.RTM. 488-QSY.RTM. 7.
[0069] As used herein, the term "donor fluorophore" means a
molecule that, when irradiated with light of a certain wavelength,
emits light, also denoted fluorescence, of a different wavelength.
The term fluorophore is synonymous in the art with the term
"fluorochrome."
[0070] The term "acceptor," as used herein, refers to a molecule
that can absorb energy from, and upon excitation of, a donor
fluorophore and is a term that encompasses fluorophores as well as
non-fluorescent molecules. An acceptor useful in a toxin substrate
has an absorbance spectrum which overlaps the emission spectrum of
a donor fluorophore. An acceptor useful in the invention generally
also has rather low absorption at a wavelength suitable for
excitation of the donor fluorophore.
[0071] When carrying out a FRET assay using the toxin substrate of
the present invention, in an embodiment the toxin substrate
contains a cleavage site that "intervenes" between a donor
fluorophore and an acceptor having an absorbance spectrum which
overlaps the emission spectrum of the donor fluorophore. Thus, the
cleavage site is positioned in between the fluorophore and acceptor
such that cleavage at the site results in a first molecule
containing the fluorophore and a second molecule containing the
acceptor. All or only a portion of the toxin recognition sequence
can intervene between the donor fluorophore and acceptor.
[0072] The present invention also provides methods of determining
toxin protease activity. Such methods are valuable, in part,
because they are amenable to rapid screening and do not require
separation of cleaved products from uncleaved substrate.
Furthermore, the methods of the present invention are used with
cells that have transfected with the toxin substrate using the
carrier described herein. Such cells can be assayed in the presence
or absence of molecules, compounds, drugs, or conditions to be
tested. The methods of the invention include the following steps:
(a) contacting a sample having cells to be assayed with the toxin
or fragment thereof, as described herein, and a lipid or polymeric
carrier (e.g., a DNA uptake facilitating agent) under conditions
suitable for toxin protease activity, wherein the toxin substrate
that contains a donor fluorophore, an acceptor having an absorbance
spectrum overlapping the emission spectrum of the donor
fluorophore, and a toxin recognition sequence containing a cleavage
site, wherein the cleavage site intervenes between the donor
fluorophore and the acceptor and wherein, under the appropriate
conditions, resonance energy transfer is exhibited between the
donor fluorophore and the acceptor; (b) exciting the donor
fluorophore; and (c) determining resonance energy transfer of the
treated substrate relative to a control substrate, where a
difference in resonance energy transfer of the treated substrate as
compared to the control substrate is indicative of protease
activity. An additional step in an embodiment of the present
invention is to subject the sample to a drug, compound or molecule
to be tested. The compound to be assayed can be at concentrations
as desired by the user carrying out the assay. In an embodiment,
the concentration of compound to be assayed is serial diluted. A
method of the invention can be practiced with an acceptor which is
a fluorophore, or with a non-fluorescent acceptor.
[0073] In a method of the invention, resonance energy transfer can
be determined by a variety of means. In one embodiment, the step of
determining resonance energy transfer includes detecting donor
fluorescence intensity of the treated substrate, wherein increased
donor fluorescence intensity of the treated substrate as compared
to the control substrate is indicative of toxin protease activity.
In another embodiment, the step of determining resonance energy
transfer includes detecting acceptor fluorescence intensity of the
treated substrate, wherein decreased acceptor fluorescence
intensity of the treated substrate as compared to the control
substrate is indicative of toxin protease activity. In a further
embodiment, the step of determining resonance energy transfer
includes detecting the acceptor emission maximum and the donor
fluorophore emission maximum, wherein a shift in emission maxima
from near an acceptor emission maximum to near a donor fluorophore
emission maximum is indicative of toxin protease activity. In an
additional embodiment, the step of determining resonance energy
transfer includes detecting the ratio of fluorescence amplitudes
near an acceptor emission maximum to fluorescence amplitudes near a
donor fluorophore emission maximum, wherein a decreased ratio in
the treated sample as compared to the control sample is indicative
of toxin protease activity. In yet a further embodiment, the step
of determining resonance energy transfer is practiced by detecting
the excited state lifetime of the donor fluorophore in the treated
substrate, wherein an increased donor fluorophore excited state
lifetime in the treated substrate as compared to the control
substrate is indicative of toxin protease activity.
[0074] As discussed further below, a variety of conditions suitable
for toxin protease activity are useful in a method of the
invention. For example, conditions suitable for toxin protease
activity can be provided such that at least 10% of the substrate is
cleaved. Similarly, conditions suitable for toxin protease activity
can be provided such that at least 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or 95% of the toxin substrate is cleaved, or such that
100% of the toxin substrate is cleaved. In one embodiment, the
conditions suitable for toxin protease activity are selected such
that the assay is linear.
[0075] As used herein, the term "sample" means any biological
matter that contains the toxin or toxin fragment, as described
herein. In an embodiment, the toxin includes light chain or
proteolytically active fragment thereof. Thus, the term sample
encompasses but is not limited to purified or partially purified
toxins; recombinant single chain or dichain toxin with a naturally
or non-naturally occurring sequence; chimeric toxin containing
structural elements from multiple toxin species or subtypes;
recombinant toxin light chain with a naturally occurring or
non-naturally occurring sequence; bulk toxin; formulated product;
cells or crude, fractionated or partially purified cell lysates,
for example, engineered to include a recombinant nucleic acid
encoding a toxin or light chain thereof, including bacterial,
baculoviral and yeast lysates.
[0076] In the methods of the invention, a sample is treated with a
toxin under conditions suitable for toxin protease activity.
Exemplary conditions suitable for toxin protease activity are well
known in the art, and further can be determined by routine methods.
See, for example, Hallis et al., J. Clin. Microbiol. 34:1934-1938
(1996); Ekong et al., Microbiol. 143:3337-3347 (1997); Shone et
al., WO 95/33850; Schmidt and Bostian, supra, 1995; Schmidt and
Bostian, supra, 1997; Schmidt et al., supra, 1998; and Schmidt and
Bostian, U.S. Pat. No. 5,965,699. It is understood that conditions
suitable for toxin protease activity can depend, in part, on the
specific toxin type or subtype being assayed and the purity of the
toxin preparation. Conditions suitable for toxin protease activity
generally include a buffer, such as HEPES, Tris or sodium
phosphate, typically in the range of pH 5.5 to 9.5, for example, in
the range of pH 6.0 to 9.0, pH 6.5 to 8.5 or pH 7.0 to 8.0.
Conditions suitable for toxin protease activity also can include,
if desired, dithiothreitol or mercaptoethanol or another reducing
agent, for example, where a dichain toxin is being assayed (Ekong
et al., supra, 1997). In one embodiment, the conditions include DTT
in the range of 0.01 mM to 50 mM; in other embodiments, the
conditions include DTT in the range of 0.1 mM to 20 mM, 1 to 20 mM,
or 5 to 10 mM. If desired, the toxin or fragment or sample can be
pre-incubated with a reducing agent, for example, with 10 mM
dithiothreitol (DTT) for about 30 minutes prior to addition of
toxin substrate. Toxins are zinc metalloproteases, and a source of
zinc, such as zinc chloride or zinc acetate, typically in the range
of about 1 to 500 .mu.M, for example, about 5 to 10 .mu.M can be
included, if desired, as part of the conditions suitable for toxin
protease activity. Zinc chelators such as EDTA generally are
excluded from a buffer for assaying toxin protease activity.
[0077] Conditions suitable for toxin protease activity also can
include, if desired, bovine serum albumin (BSA). When included, BSA
typically is provided in the range of 0.1 mg/ml to 10 mg/ml. In one
embodiment, BSA is included at a concentration of 1 mg/ml. See, for
example, Schmidt and Bostian, supra, 1997.
[0078] The amount of toxin or fragment thereof can be varied in a
method of the invention. Peptide substrate concentrations useful in
a method of the invention include concentrations, for example, in
the range of 5 .mu.M to 3.0 mM. A peptide substrate can be supplied
at a concentration, for example, of 5 .mu.M to 500 .mu.M, 5 .mu.M
to 50 .mu.M, 50 .mu.M to 3.0 mM, 0.5 mM to 3.0 mM, 0.5 mM to 2.0
mM, or 0.5 mM to 1.0 mM. The skilled artisan understands that the
concentration of toxin substrate or the amount of sample can be
limited, if desired, such that the assay is linear. At increasingly
high concentrations of substrate or toxin, linearity of the assay
is lost due to the "inner filter effect," which involves
intermolecular energy transfer. Thus, in one embodiment, a method
of the invention relies on a toxin substrate concentration which is
limited such that intermolecular quenching does not occur. In
another embodiment, a method of the invention relies on a toxin
substrate concentration of less than 100 .mu.M. In further
embodiments, a method of the invention relies on a toxin substrate
concentration of less than 50 .mu.M or less than 25 .mu.M. If
desired, a linear assay also can be performed by mixing toxin
substrate with corresponding, "unlabeled" substrate which lacks the
donor fluorophore and acceptor of the toxin substrate. The
appropriate dilution can be determined, for example, by preparing
serial dilutions of toxin substrate in the corresponding unlabeled
substrate.
[0079] The concentration of purified or partially purified toxin or
fragment thereof assayed in a method of the invention generally is
in the range of about 0.0001 to 5000 ng/ml toxin, for example,
about 0.001 to 5000 ng/ml, 0.01 to 5000 ng/ml, 0.1 to 5000 ng/ml, 1
to 5000 ng/ml, or 10 to 5000 ng/ml toxin, which can be, for
example, purified recombinant light chain or dichain toxin or
formulated toxin product containing human serum albumin and
excipients. Generally, the amount of purified toxin used in a
method of the invention is in the range of 0.1 pg to 10 .mu.g.
Purified, partially purified or crude samples can be diluted to
within a convenient range for assaying for toxin protease activity
against a standard curve. Similarly, a sample can be diluted, if
desired, such that the assay for toxin protease activity is
linear.
[0080] Conditions suitable for toxin protease activity also
generally include, for example, temperatures in the range of about
20.degree. C. to about 45.degree. C., for example, in the range of
25.degree. C. to 40.degree. C., or the range of 35.degree. C. to
39.degree. C. Assay volumes often are in the range of about 5 to
about 200 .mu.l, for example, in the range of about 10 .mu.l to 100
.mu.l or about 0.5 .mu.l to 100 .mu.l, although nanoliter reaction
volumes also can be used with the methods of the invention. Assay
volumes also can be, for example, in the range of 100 .mu.l to 2.0
ml or in the range of 0.5 ml to 1.0 ml.
[0081] Assay times can be varied as appropriate by the skilled
artisan and generally depend, in part, on the concentration, purity
and activity of the toxin. In particular embodiments, at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the toxin
substrate is cleaved. In further embodiments, the protease reaction
is stopped before more than 5%, 10%, 15%, 20%, 25% or 50% of the
toxin substrate is cleaved. Protease reactions can be terminated,
for example, by addition of H.sub.2SO.sub.4 as, addition of about
0.5 to 1.0 sodium borate, pH 9.0 to 9.5, or addition of zinc
chelators. One skilled in the art understands that protease
reactions can be terminated prior to exciting the donor fluorophore
or determining energy transfer.
[0082] Proteolysis of the toxin substrate, and hence toxin protease
activity, can be detected by a variety of means, for example, by
detecting an increased donor fluorescence intensity; a decreased
acceptor fluorescence intensity; a shift in emission maxima from
near the acceptor emission maximum to near the donor fluorophore
emission maximum; a decreased ratio of fluorescence amplitudes near
the acceptor emission maximum to the fluorescence amplitudes near
the donor fluorophore emission maximum; or an increased donor
fluorophore excited state lifetime. The relevant fluorescence
intensities or excited state lifetimes are detected at the
appropriate selected wavelength or range of wavelengths. For
example, where donor fluorescence intensity is detected, the
appropriate selected wavelength at or near the emission maxima of
the donor fluorophore, or a range of wavelengths encompassing or
near to the emission maxima of the donor fluorophore. See
20080038756 for a discussion of carrying out FRET assays for
certain toxins.
Endosomal Release:
[0083] An insulin release assay can be performed to determine the
extent intoxication. In response to glucose stimulation, an
insulinoma cell line, e.g., HIT-T15 secretes insulin in an exocytic
process that depends on the activity of SNAP-25 for vesicle docking
and fusion. If insulinoma cells lack a toxin receptor, these cells
would be unable to uptake toxin upon exposure to this toxin and
insulin secretion could occur in the presence of high glucose in
the media. However, if insulinoma cells contain a toxin receptor,
insulin secretion would be inhibited after toxin treatment since
the toxin could intoxicate the cell and cleave SNAP-25.
[0084] To conduct an inhibition assay for insulin release, a
suitable density of cells such as HIT-T15 cells is plated and grown
according to conditions known in the art for growing and
maintaining cell lines. The toxin or fragment along with a
polymeric or lipid carrier, as described herein, is then contacted
with the cells under conditions suitable for intoxication of the
cells with the toxin or fragment. The cells are subjected to
glucose, in varying concentrations (e.g., ranging from 4.0 mM
glucose (low) to about 30 mM glucose (high)). Incubating the cells
at an appropriate temperature (e.g., about 37.degree. C.) allows
insulin secretion to occur in cells into which the toxin or
fragment has entered and wherein binding of the substrate to a
receptor occurs. Alternatively intoxication can be monitored by
blockage of vesicle mediated secretion after depolization with 5 mM
potassium solution. In the case in which a compound, drug, molecule
or condition prevents such binding (e.g., an antagonist), insulin
secretion is also inhibited. In the case in which the compound,
drug, molecule or condition allows or is an agonist of such
binding, insulin secretion occurs or is increased. The amount of
insulin present in the condition media samples was determined can
be determined using an insulin ELISA assay (Peninsula Laboratories,
Inc., San Carlos, Calif.). With this particular assay, e.g.,
exocytosis is expressed as the amount of insulin secreted per
1.5.times.10.sup.5 cell/hr. Any method known in the art for
detecting insulin or glucose can be used. Another approach to using
HIT-T15 cells is to transfect the cells with a secreted peptide
reporter such as luciferase fused to proinsulin or human growth
hormone and assaying for release of the peptide transmitter after
depolarization with potassium or stimulation with high glucose
containing media.
Systems and Kits
[0085] The present invention further relates to systems and kits.
The system or kit includes, e.g., one or more cell lines, one or
more lipid or polymeric carriers (e.g., a DNA uptake facilitating
agent), and/or one or more toxins or fragments thereof, as defined
herein. The cell line, in embodiments, can already be intoxicated
with the toxin or fragment and is therefore ready for cell based
assays, e.g., to be evaluated by a drug candidate.
Polypeptides, Nucleic Acid Sequences, Vectors, Host Cells of the
Present Invention
[0086] As used herein, the term "recombinant" refers to a molecule
that is one that is genetically made using techniques described
herein.
[0087] As used herein, the term "polypeptide" encompasses amino
acid chains of the toxin having any length, partial or full length
proteins, wherein the amino acid residues are linked by covalent
peptide bonds. Thus, a polypeptide can comprise a portion of the
toxin or domain thereof, such as heavy chains, light chains and
combinations thereof.
[0088] The polypeptides of the present invention referred to herein
as "isolated" are polypeptides that are separated away from other
proteins and cellular material of their source of origin. The
compositions and methods of the present invention also encompass
variants of polypeptides and DNA molecules of the present
invention. A polypeptide "variant," as used herein, is a
polypeptide that differs from the recited polypeptide only in
conservative substitutions and/or modifications, such that the
ability of the toxin is retained.
[0089] The present invention also encompasses proteins and
polypeptides, variants thereof, or those having amino acid
sequences analogous to the amino acid sequences of binding agents
described herein. Such polypeptides are defined herein as analogs
(e.g., homologues), or mutants or derivatives. "Analogous" or
"homologous" amino acid sequences refer to amino acid sequences
with sufficient identity of any one of the amino acid sequences of
the present invention so as to possess the biological activity
(e.g., the ability to bind to the toxin). For example, an analog
polypeptide can be produced with "silent" changes in the amino acid
sequence wherein one, or more, amino acid residues differ from the
amino acid residues of any one of the sequence, yet still possesses
the function or biological activity of the polypeptide. In
particular, the present invention relates to homologous polypeptide
molecules having at least about 40% (e.g., 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90% or 95%) identity or similarity with SEQ ID
NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 or combination
thereof. Percent "identity" refers to the amount of identical
nucleotides or amino acids between two nucleotides or amino acid
sequences, respectfully. As used herein, "percent similarity"
refers to the amount of similar or conservative amino acids between
two amino acid sequences.
[0090] Homologous polypeptides can be determined using methods
known to those of skill in the art. Initial homology searches can
be performed at NCBI against the GenBank, EMBL and SwissProt
databases using, for example, the BLAST network service.
Altschuler, S. F., et al., J. Mol. Biol., 215:403 (1990),
Altschuler, S. F., Nucleic Acids Res., 25:3389-3402 (1998).
Computer analysis of nucleotide sequences can be performed using
the MOTIFS and the FindPatterns subroutines of the Genetics
Computing Group (GCG, version 8.0) software. Protein and/or
nucleotide comparisons were performed according to Higgins and
Sharp (Higgins, D. G. and Sharp, P. M., Gene, 73:237-244 (1988)
e.g., using default parameters).
[0091] The present invention, in one embodiment, includes an
isolated nucleic acid molecule having a sequence of SEQ ID NO: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 or combinations thereof. As
used herein, the terms "DNA molecule" or "nucleic acid molecule"
include both sense and anti-sense strands, cDNA, genomic DNA,
recombinant DNA, RNA, and wholly or partially synthesized nucleic
acid molecules. A nucleotide "variant" is a sequence that differs
from the recited nucleotide sequence in having one or more
nucleotide deletions, truncations, substitutions or additions. Such
modifications can be readily introduced using standard mutagenesis
techniques, such as oligonucleotide-directed site-specific
mutagenesis as taught, for example, by Adelman et al. (DNA 2:183,
1983). Nucleotide variants can be naturally occurring allelic
variants, or non-naturally occurring variants. Variant nucleotide
sequences preferably exhibit at least about 70%, more preferably at
least about 80% and most preferably at least about 90% homology to
the recited sequence. Such variant nucleotide sequences will
generally hybridize to the recited nucleotide sequence under
stringent conditions. In one embodiment, "stringent conditions"
refers to prewashing in a solution of 6.times.SSC, 0.2% SDS;
hybridizing at 65.degree. Celsius, 6.times.SSC, 0.2% SDS overnight;
followed by two washes of 30 minutes each in 1.times.SSC, 0.1% SDS
at 65.degree. C. and two washes of 30 minutes each in
0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0092] The present invention also encompasses isolated nucleic acid
sequences that encode the polypeptide and in particular, those
which encode a polypeptide molecule having an amino acid sequence
of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22 or
combinations thereof.
[0093] As used herein, an "isolated" nucleotide sequence is a
sequence that is not flanked by nucleotide sequences which normally
(e.g., in nature) flank the gene or nucleotide sequence (e.g., as
in genomic sequences) and/or has been completely or partially
purified from other transcribed sequences (e.g., as in a cDNA or
RNA library). Thus, an isolated gene or nucleotide sequence can
include a gene or nucleotide sequence which is synthesized
chemically or by recombinant means. Nucleic acid constructs
contained in a vector are included in the definition of "isolated"
as used herein. Also, isolated nucleotide sequences include
recombinant nucleic acid molecules and heterologous host cells, as
well as partially or substantially or purified nucleic acid
molecules in solution. The nucleic acid sequences that encode the
toxin of the present invention include homologous nucleic acid
sequences. "Analogous" or "homologous" nucleic acid sequences refer
to nucleic acid sequences with sufficient identity of any one of
the nucleic acid sequences described herein, such that once encoded
into polypeptides, they possess the biological activity of any one
of the toxins herein. In particular, the present invention is
directed to nucleic acid molecules having at least about 40% (e.g.,
45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) identity
with SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 or
combinations thereof.
[0094] Also encompassed by the present invention are nucleic acid
sequences, DNA or RNA, which are substantially complementary to the
DNA sequences encoding the polypeptides of the present invention,
and which specifically hybridize with their DNA sequences under
conditions of stringency known to those of skill in the art. As
defined herein, substantially complementary means that the nucleic
acid need not reflect the exact sequence of the sequences, but must
be sufficiently similar in sequence to permit hybridization with
nucleic acid sequence under high stringency conditions. For
example, non-complementary bases can be interspersed in a
nucleotide sequence, or the sequences can be longer or shorter than
the nucleic acid sequence, provided that the sequence has a
sufficient number of bases complementary to the sequence to allow
hybridization therewith. Conditions for stringency are described in
e.g., Ausubel, F. M., et al., Current Protocols in Molecular
Biology, (Current Protocol, 1994), and Brown, et al., Nature,
366:575 (1993); and further defined in conjunction with certain
assays.
Stringency Conditions for Nucleic Acids:
[0095] Specific hybridization can be detected under high stringency
conditions. "Stringency conditions" for hybridization is a term of
art which refers to the conditions of temperature and buffer
concentration which permit and maintain hybridization of a
particular nucleic acid to a second nucleic acid; the first nucleic
acid may be perfectly complementary to the second, or the first and
second may share some degree of complementarity which is less than
perfect. For example, certain high stringency conditions can be
used which distinguish perfectly complementary nucleic acids from
those of less complementarity. "High stringency conditions" for
nucleic acid hybridizations and subsequent washes are explained,
e.g., on pages 2.10.1-2.10.16 and pages 6.3.1-6 in Current
Protocols in Molecular Biology (Ausubel, et al., In: Current
Protocols in Molecular Biology, John Wiley & Sons, (1998)). The
exact conditions which determine the stringency of hybridization
depend not only on ionic strength, temperature and the
concentration of destabilizing agents such as formamide, but also
on factors such as the length of the nucleic acid sequence, base
composition, percent mismatch between hybridizing sequences and the
frequency of occurrence of subsets of that sequence within other
non-identical sequences. Thus, high stringency conditions can be
determined empirically.
[0096] By varying hybridization conditions from a level of
stringency at which no hybridization occurs to a level at which
hybridization is first observed, conditions which will allow a
given sequence to hybridize (e.g., selectively) with the most
similar sequences in the sample can be determined. Exemplary
conditions are described in the art (Krause, M. H., et al., 1991,
Methods Enzymol. 200:546-556). Also, low and moderate stringency
conditions for washes are described (Ausubel, et al., In: Current
Protocols in Molecular Biology, John Wiley & Sons, (1998)).
Washing is the step in which conditions are usually set so as to
determine a minimum level of complementarity of the hybrids.
Generally, starting from the lowest temperature at which only
homologous hybridization occurs, each .degree. C. by which the
final wash temperature is reduced (holding SSC concentration
constant) allows an increase by 1% in the maximum extent of
mismatching among the sequences that hybridize. Generally, doubling
the concentration of SSC results in an increase in Tm of about
17.degree. C. Using these guidelines, the washing temperature can
be determined empirically for high stringency, depending on the
level of the mismatch sought. In some embodiments, high stringency
conditions include those in which nucleic acid with less than a few
mismatches does not bind. High stringency conditions, using these
guidelines, lie in a temperature range between about 40.degree. C.
and about 60.degree. C., an SSC concentration range between about
1.times. and about 10.times. (e.g., about 2.times.), and a reaction
time range of between about 30 seconds and about 36 hours.
EXEMPLIFICATION
Example 1
Summary
[0097] The present invention improves the utility of cell-based
toxin assays through the use of a novel toxin delivery system that
easily and dramatically increases the functional entry of botulinum
neurotoxin (BoNT), and probably other related toxins, into cells.
It was discovered that the functional delivery of botulinum
neurotoxin (BoNT), serotype A, to cultured neuronal cells can be
substantially improved by combining it with DNA transfection
reagents before application. Surprisingly, this toxin
"transduction" approach is also successful for the delivery of BoNT
to all non-neuronal cells tested, cells which normally are
completely refractory to intoxication. Finally, intoxication
efficiencies were achieved approaching those of holotoxin in these
cell lines by transducing only the BoNT catalytic domain into the
cells using the DNA transfection reagents, obviating the need to
use holotoxin to intoxicate cultured cells and avoiding the
consequent safety and regulatory issues. This methodology improves
functional cell delivery of other toxins, particularly those that
normally intoxicate cells using cell entry mechanisms similar to
BoNT. This invention also facilitates the testing of the functional
consequences of mutations in BoNT LC as mutated versions of the
Light Chain (LC) because recombinant proteins can be delivered to
cells at physiologically relevant levels. This invention thus
permits a broad expansion of the available cell and toxin options
for the development of cell-based intoxication assays and improves
the ability to screen for therapeutic agents that prevent or
reverse toxin pathologies.
Example 2
Methods
Materials and Methods.
Cell Culture and Reagents:
[0098] M17 (ATCC# CRL-2267) cells were maintained in DMEM (Gibco,
USA) containing 10% fetal bovine serum (FBS) (Gibco, USA). MEME
(Gibco, USA) plus 10% FBS media were used for culturing Neuro2a
(ATCC# CCL-131) and HEK293 (ATCC# CRL-1573) cells. HIT-T15 (ATCC#
CRL-1777) cells were cultured in F12K (Gibco, USA) containing 10%
horse and 5% FBS. 6.times.10.sup.4 cells were seeded onto each well
of 24-well plate and maintained at 37.degree. C. After 72 hrs,
culture medium was replaced with fresh medium before experimental
treatments. Primary cultures of cerebellar granule cells were
prepared from 7 day-old Sprague-Dawley rats essentially by the
methods of Farkas. Briefly, after aseptically removing cerebella
from the skulls, tissue was freed from meninges and incubated in
0.05% trypsin solution for 10 min at RT. After a brief
centrifugation, cells were triturated in DMEM/F12 containing 10%
FBS and filtered through a sterile cell strainer mesh with 40 um
pore size (BD Falcon, USA). Cell number was determined by trypan
blue exclusion, and cells were seeded onto poly-L-lysine (PLL) 1
g/cm2 laminin (Sigma, USA) coated 6 well plate with DMEM containing
10% FBS, 25 mM KCl, 2 mM Glutamax, and 100 g/mL gentamicin (Gibco,
USA). The cultures were maintained at 37.degree. C. in a humidified
atmosphere of 6% CO2. After 24 hr of culturing cytosine arabinoside
(Sigma, USA) was added to a final concentration of 20 .mu.M to
prevent astrocytic proliferation. The neurons were cultured for 7-8
days before use. FuGene-HD (Roche, USA), Lipofectamine 2000
(Invitrogen, USA) and PEI average molecular weights 0.6, 1.8, 10,
70 kDa (Alfa Aesar, USA) and 25 kDa (Sigma, USA) of various average
molecular weights (Sigma-Aldrich) were used for transfection and
transduction as recommended by the manufacturer except where
indicated. Bafilomycin A1 was obtained from Tocris Cookson Inc
(USA). BoNT/A (isotype 1), BoNT/B and BoNT/E were obtained from
Metabiologics.
[0099] Resources: Recombinant BoNT/B light chain protease (amino
acids 1-441) was expressed in pET14b with hexahistidine tags at the
amino and carboxyl termini. The protein was expressed within E.
coli in soluble form and purified to near homogeneity by standard
nickel affinity chromatography. The recombinant BoNT/E LC was
expressed in E. coli as a N-terminal fusion protein with
glutathione-S-transferase. The protein was purified by standard
glutathione affinity methods and provided as a gift by Dr. Randall
Kincaid (Veritas Labs, USA). Antibodies: rabbit anti-SNAP25
antibody (Sigma); goat anti-rabbit HRP antiserum (Sigma); rabbit
anti-VAMP2 antibody (Millipore); rabbit anti-CFP (gift from Dr.
Randall Kincaid, Veritas Labs). Reagents for Western blotting
including Wash Solution and LumiGLO Chemiluminescent Substrate were
purchased from (KPL, USA).
BoNT/A Holotoxin Intoxication and Transduction:
[0100] Neuronal cell lines M17, Neuro2a and non-neuronal cell lines
HEK293, HIT-T15 were tested in transfection reagent facilitated
intoxication. 50 .mu.l of serum free medium was used to prepare
toxin dilution or toxin transfection mixture for each well of a
24-well plate. 0.75 .mu.g BoNT/A was added with or without
transfection reagent with a ratio of 1:3 (toxin [.mu.g]:
transfection reagent [.mu.l]) and incubated at room temperature for
more than 15 min. Apply the transfection mixture onto each wells. 3
hrs and/or 24 hrs after transfection, cells were washed twice with
1 ml DPBS and incubated with fresh medium. Cell extracts were
prepared 48 h post transfection. Cells were washed once with 1 ml
DPBS, trypsinized, and washed once with 1 ml DPBS. 50 .mu.l of
sample buffer plus beta-mercaptoethanol was added to lyse cells and
protein samples were boiled for 10 min. Toxin transduction
efficiency was measured indirectly by monitoring the endogenous
SNAP25 cleavage.
[0101] For testing toxin delivery efficacy through commercially
available transfection reagents, Neuro2a cells were exposed to a
toxin-transfection reagent mixture for various durations. Briefly,
50 .mu.l serum free medium was used for each well of 24-well plate.
BoNT/A was added with FuGene-HD using 3 .mu.l per .mu.g of toxin
except where indicated. A well treated with toxin alone without
FuGene-HD was included as a control. Apply the mixture onto each
well. 0.5, 1, 2, 3, 6 and 24 hrs after transfection, cells were
lysed with sample buffer plus beta-mercaptoethanol immediately or
washed twice with 1 ml DPBS and incubated with fresh medium. Cell
extracts were prepared 48 hrs post transduction with the procedures
noted above. Toxin transduction efficiency was checked indirectly
by monitoring the endogenous SNAP25 cleavage.
[0102] For testing the effective range of concentrations for
transfection reagent-mediated cell intoxication, BoNT/A
concentrations ranging from 0.1 to 10 nM were used in Neuro2a
cells. Similar transduction procedures were taken with some
modifications. 7.5 ng, 75 ng and 0.75 .mu.g BoNT/A was diluted to
50 .mu.l serum free medium followed by addition of transfection
reagent with a ratio of 1:3 (toxin [.mu.g]: transfection reagent
[.mu.l]) and incubated in room temperature for more than 15 min.
FuGene-HD left out of toxin dilution was designated as control. 3
hrs and 24 hrs after transfection, cells were washed twice with 1
ml DPBS and incubated with fresh medium. Cell extracts were
prepared 48 h post transduction. Toxin transduction efficiency is
checked indirectly by monitoring the endogenous SNAP25
cleavage.
BoNT/A Lc Protease Transduction:
[0103] The neuronal cell lines M17, Neuro2a and the non-neuronal
cell lines HEK293, HIT-T15 were tested in transfection reagent
facilitated Lc transduction. 50 .mu.l serum free medium was used
for preparing Lc dilution or Lc transduction mixture for each well
of 24-well plate. 0.75 .mu.g Lc438 or Lc424 was added with or
without transfection reagent with a ratio of 1:3 (Lc [.mu.g]:
transfection reagent [.mu.l]) and incubated in room temperature for
more than 15 min. Apply the transfection mixture onto each wells.
24 hrs after transfection, cell extracts were prepared with the
procedures noted above. Toxin transfection efficiency was checked
indirectly by monitoring the endogenous SNAP25 cleavage.
BoNT/E Intoxication and Transduction
[0104] BoNT/E holotoxin was activated before cell intoxication. 1
mg/ml BoNT/E was nicked by incubating for 30 min at 37.degree. C.
with 0.3 mg/ml trypsin (type XI, bovine pancreas) in 30 mM HEPES,
pH 6.75. Trypsin was subsequently inhibited by addition of 0.5
mg/ml trypsin inhibitor (type I-S, soybean) and incubation for 15
min at 20.degree. C.
[0105] Toxins were aliquoted and stored at -20.degree. C.; each
experiment utilized a new aliquot of toxin to ensure uniform
activity. Neuronal cell lines M17 and Neuro2a cell lines were
tested in transfection reagent facilitated intoxication with
procedures noted in 2.3.
Cell Viability Assay
[0106] MTT assays were performed to measure cell viability. M17 and
Neuro2a cells were seeded onto 96 well plate for 24 hrs and cells
were treated with recombinant Lc with or without preincubation with
transfection reagents for another 24 hrs. Cell viability was
measured 24 hrs after transduction and experimental procedures were
followed according to manufacture instructions. Record absorbance
at 570 nM with Synergy.TM. HT Multi-Mode Microplate Reader, and
data were analyzed with KC4 software. Experiments were performed in
triplicate.
Bafilomycin Blockage of BoNT/A Nature Intoxication:
[0107] The neuronal cell lines M17, Neuro2a and non-neuronal cell
lines HEK293, HIT-T15 were treated with 1 .mu.M of Bafilomycin A1
or DMSO for 2 hrs and washed with 1 ml DPBS twice before
transduction. 0.75 .mu.g BoNT/A or Lc438 was transduced to cell
lines with the same preparation noted above. Due to the toxin
insensitivity of neuroblastoma cell lines, primary cells were
incorporated in this experiment as control to show the bafilomycin
drug effect on BoNT intoxication in the absence of commercial DNA
transfection reagent. For primary cells, 37.5 ng BoNT/A was added
with or without transfection reagent. 4 hrs and 24 hrs after
transfection, cell extracts were prepared as above. The inhibitory
effect of Bafilomycin onto toxin/Lc was measured indirectly by
monitoring the endogenous SNAP25 cleavage.
Bafilomycin Effect on DNA Transfection:
[0108] 0.5 .mu.g CFP plasmid was transfected to M17, Neuro2a and
HIT-T15 cells pre-treated with bafilomycin A1 for 2 hrs. 3 hrs or
24 hrs after transfection, cells were washed twice with 1 ml DPBS
and incubated with fresh medium. 24 hrs post transfection
fluorescence signals were recorded before cell extracts were
prepared with the same procedures noted above. The inhibitory
effect of bafilomycin onto DNA transfection was measured by
monitoring the cell fluorescence signals through Olympus IX50
microscope and the CFP protein expression through Western
Blotting.
Western Blotting:
[0109] Cell extract prepared from 4.times.10.sup.5 cells was boiled
for 5 min and loaded to 15% pre-casted protein gels (BioRad).
Protein samples were separated and transferred to PVDF membrane
with cool temperature. To eliminate non-specific binding blots were
incubated with 5% skim milk/PBST 0.5% for at least 1 hr at room
temperature. Blots were incubated with primary antibodies: Rabbit
anti SNAP25 (1:5000), Rabbit anti XFP (1:5000), Rabbit anti VAMP2
(1:5000) at 4.degree. C. overnight. Blots were then washed with
PBST 0.5% buffer and then treated with goat anti rabbit antibody
(1:5000) with HRP label was subsequently incubated with blots for 1
hr at room temperature before signal detection using LumiGLO
Chemiluminescent Substrate (KPL). Signals were scanned by Kodak
[instruments] and analyzed with the Kodak 1D 3.6 network.
Example 3
Results
Commercial Lipid-Based DNA Transfection Reagents Enhance Botulinum
Intoxication of Cultured Neuronal Cells.
[0110] It was observed that neuronal cells intoxicated with BoNT/A
immediately after DNA transfection using the FuGene-HD reagent
(Roche) appeared more efficiently intoxicated than control cells so
the effect of FuGene-HD on BoNT intoxication was directly tested.
The measure of BoNT serotype A intoxication used in these studies
was the percentage of the cellular SNAP25 that had been cleaved. As
shown in FIG. 1, virtually no SNAP25 was cleaved following 3 hrs
intoxication and about 50% was cleaved with 24 hrs exposure to
BoNT/A in M17. When the toxin was pre-incubated with FuGene-HD
prior to addition to media, approximately 50% of SNAP25 became
cleaved with 3 hrs of toxin exposure and 90% was cleaved in M17
after a 24 hrs exposure. The effect of the FuGene-HD DNA
transfection reagent on intoxication efficiency was even more
profound in the Neuro2a neuroblastoma cell line (FIG. 1). Under
normal intoxication conditions, no cleavage of SNAP25 was detected
following a 3 hrs exposure to toxin and about 10% cleavage occurred
with a 24 hr toxin exposure. When intoxication was performed
following preincubation with FuGene-HD, about 80% and 95% of
cellular SNAP25 was cleaved with 3 hr and 24 hr toxin transfection
respectively. Enhancement of intoxication also occurred when the
toxin and FuGene-HD were separately added to neuronal cells, but
the effect was smaller (not shown). Thus, the presence of FuGene-HD
accelerated the efficiency of intoxication following short exposure
and increased the level of intoxication following a long exposure.
In the case of Neuro2a, the presence of FuGene-HD converted a cell
line from one that was poorly intoxicated by BoNT/A to one that is
very efficiently intoxicated. The amount of enhanced intoxication
did not change significantly using several fold more or less
FuGene-HD and the ratio of 1:3 was selected for routine use to
minimize toxicity to the cells. Nevertheless, to exclude the
possibility that transfection reagents increased cell death leading
to release of SNAP25 and enhanced cleavage, MTT assays were
performed and revealed no significant change in cell viability
following exposure to FuGene-HD at 1:3 (data not shown).
[0111] To characterize the rate at which DNA transfection
reagent-enhanced intoxication occurs, Neuro2a cells were exposed to
BoNT/A in the presence of FuGene-HD for variable times and
harvested immediately. As shown in FIG. 2, cleavage of SNAP25 could
be observed in as little as 30 minutes and maximum cleavage of
about 90% occurred within the first 6 hrs. In this experiment, the
Neuro2a cells were not detectably intoxicated in the absence of
FuGene-HD. When the medium was changed and the cells were cultured
for an additional 2 days after short toxin exposures (1-3 hrs) but
not longer exposures (6 or 24 hrs), a small amount of additional
SNAP25 cleavage occurred but never reached the 90% cleavage
observed after only 6 hours exposure to toxin (data not shown).
This suggests that the BoNT protease rapidly reaches the cell
cytosol and most SNAP25 cleavage is complete within only a few
hours of the toxin entering the cell. These results also suggest
that the uncleaved SNAP25 observed in these experiments is from
cells that have not become intoxicated.
[0112] Typically neuroblastoma cells require BoNT concentrations of
10 nM or more to detect significant intoxication while primary
neurons are often sensitive to BoNT concentrations as low as 100
pM. Experiments were performed to examine the sensitivity of DNA
transfection reagent enhanced intoxication of neuroblastoma cells
to toxin concentrations. Neuro2a cells were cultured for 24 hrs
with BoNT/A concentrations ranging from 0.1 to 10 nM (FIG. 3) and
harvested 48 hrs after toxin exposure. In the absence of FuGene-HD,
the Neuro2a cells were not detectably intoxicated even at 10 nM. In
the presence of FuGene-HD, some cleavage of SNAP25 was observed in
Neuro2a cells exposed to as little as 100 pM BoNT/A. In a similar
experiment with only 3 hrs BoNT/A exposure, 10% and 80% of SNAP25
cleavage was detected with toxin concentrations of 1 nM and 10 nM
respectively (data not shown). Interestingly, the efficiency of
intoxication of primary neurons was not enhanced with DNA
transfection reagents (see below), suggesting that these cells are
being intoxicated with maximal sensitivity under normal conditions.
These data show that it is possible to achieve intoxication
efficiencies in at least some neuroblastoma cells that approach
those of primary neurons by including FuGene-HD with the toxin.
Commercial DNA Transfection Reagents Permit BoNT/A Intoxication of
Non-Neuronal Cells.
[0113] Results from the previous section indicated that inclusion
of commercial DNA transfection reagents during BoNT intoxication of
cells substantially increased intoxication efficiency in two
neuroblastoma cell lines. In normal intoxication, it has been shown
that botulinum toxin is internalized through receptor-mediated
endocytosis. BoNT/A uptake into cells normally requires the
presence of both ganglioside and the SV2 protein receptors which
leads to its specificity for neuronal cells. To determine whether
the use of DNA transfection reagents might bypass the need for
surface receptors, transfection reagent enhancement of intoxication
was tested in two non-neuronal cell lines that express SNAP25 yet
are not normally susceptible to BoNT/A intoxication. One cell line,
HEK293, is an embryonic kidney line and the other cell line,
HIT-T15 is a human insulinoma cell line. As expected, SNAP25
cleavage was not detected in HEK293 or HIT-T15 cells following
incubation with 10 nM of BoNT/A (FIG. 4). In contrast, inclusion of
FuGene-HD clearly facilitated the uptake of BoNT/A into these cells
leading to significant SNAP25 cleavage.
[0114] A second DNA transfection reagent, Lipofectamine 2000
(Invitrogen), was compared to FuGene-HD for the ability to enhance
BoNT/A intoxication of neuronal and non-neuronal cell lines (FIG.
4). In the case of the M17 and Neuro2a cells, both DNA transfection
reagents significantly enhanced BoNT/A uptake into cells with
FuGene-HD showing a slightly more pronounced effect. Interestingly,
the Lipofectamine 2000 reagent proved to be the more effective
reagent for enhancing BoNT uptake into HIT-T15 cells. The results
demonstrate that both lipid-based commercial DNA transfection
reagents facilitate the uptake of BoNT/A into both neuronal and
non-neuronal cells and suggest that different transfection reagents
are variably efficient in this ability when used during
intoxication of different cell lines.
Commercial DNA Transfection Reagents Facilitate Transduction of the
BoNT/A Light Chain Protease in the Absence of the BoNT/A Heavy
Chain.
[0115] It has been shown that the BoNT heavy chain (Hc) domain has
been shown to play at least two critical roles during neuronal cell
intoxication; binding to the neuronal cell receptors and
chaperoning the translocation of the BoNT light chain (Lc) protease
from the endosome to the cytosol Whether the BoNT Hc domain was
necessary for BoNT uptake facilitated by commercial DNA
transfection reagents was tested. For these experiments, two
different forms of the BoNT/A Lc were employed. The Lc438 form
contains the full size protease released following proteolytic
cleavage from the Hc domain during natural processing by the
Clostridium botulinum microbe. Lc424 is identical to Lc438 except
that 16 amino acids are removed from the carboxyl end, a
modification that does not significantly affect proteolytic
activity but improves expression and solubility properties. The
results shown in FIG. 5 show that the FuGene-HD and Lipofectamine
2000 DNA transfection reagents are efficiently able to promote the
transduction of recombinant BoNT/A Lc into both neuronal and
non-neuronal cells. Surprisingly, a very low concentration of
BoNT/A Lc was sufficient to promote internalization and cleavage of
cytosolic SNAP25, a concentration similar to that needed for
intoxication by BoNT holotoxin. Both FuGene-HD and Lipofectamine
2000 were effective in all four cell types tested, although as with
BoNT holotoxin, FuGene-HD was more effective for Neuro2a, M17 and
HEK293 while Lipofectamine 2000 was more effective with HIT-T15
cells (FIG. 4). As expected, no SNAP25 cleavage occurred in cells
when the Lc was added to the medium in the absence of DNA
transfection reagents.
Polyethyleneimine Polymers Facilitate Transduction of BoNT/A
Holotoxin and Recombinant BoNT/A Lc Protease.
[0116] The chemical nature of the commercial DNA transfection
reagents, FuGene-HD and Lipofectamine 2000, is proprietary although
both are described as lipid-based and Lipofectamine 2000 as a
cationic lipid reagent (InVitrogGen). BoNT/A toxin transduction
efficacy was tested using various polymer lengths of cationic
polyethyleneimine as a DNA transfection reagent with defined
chemical structure. The data shown in FIG. 6 demonstrate that PEI
polymers also have the ability to promote BoNT holotoxin and Lc
transduction into different cell types. The efficiency of
transduction varied widely with the use of different size PEI
polymers.
Lipid-Based Transduction of BoNT/A Lc Protease is Sensitive to
Inhibitors of ER Acidification.
[0117] During normal neuronal cell intoxication, BoNT is
internalized by receptor mediated endocytosis after which it
becomes trapped inside an endosome. Following acidification of the
endosome, the BoNT Lc protease undergoes a conformational change
and is translocated to the cytosol through a channel created by the
BoNT Hc domain. Bafilomycin is an inhibitor of vacuolar adenosine
triphosphatase and prevents endosome acidification. Previous
studies have shown that nerve cell intoxication by BoNT is
inhibited by bafilomycin. To explore the role of endosome
acidification in the DNA transfection reagent transduction of
BoNT/A Lc, transduction was performed in the presence or absence of
bafilomycin. In these experiments, cells were pre-treated with 1
.mu.M of bafilomycin A1 or DMSO for 2 hrs before transfection or
intoxication. Cytosolic internalization of the Lc was assessed by
monitoring cleavage of SNAP25 within cells. Lc internalization was
tested, in most cases, following either 4 or 24 hrs incubation with
BoNT/A holotoxin or purified Lc, plus or minus bafilomycin.
[0118] As a positive control for these experiments, pretreatment
with bafilomycin was shown to completely inhibit 4 hr BoNT/A
intoxication of primary rat granule cerebellar neurons (RGCN)
whether DNA transfection reagents were included or not (FIG. 6).
With 24 hr BoNT/A intoxication, a small amount of SNAP25 cleavage
was observed in primary cells treated with bafilomycin. Some
cytoxicity due to bafilomycin treatment was visually apparent and
so some SNAP25 cleavage may occur as BoNT/A in the media gains
access to SNAP25 released following cell lysis. Alternatively, the
effect of bafilomycin may be lost during the longer intoxication
period to permit entry of some Lc protease into the cell cytosol.
When bafilomycin was omitted, BoNT/A treatment led to nearly
complete cleavage of SNAP25. DNA transfection reagents did not
promote improved BoNT/A or BoNT/A Lc intoxication of primary
neurons (FIG. 7). These neurons were also not susceptible to
plasmid DNA transfection with these same reagents (data not
shown).
[0119] Bafilomycin inhibited SNAP25 cleavage following 4 hrs or 24
hrs incubation of both M17 and Neuro2a neuroblastoma cells with
BoNT/A holotoxin, whether intoxication was enhanced by DNA
transfection reagents or not (FIG. 7). This suggests that the
enhanced intoxication obtained with these reagents occurs through
the natural intoxication pathway including translocation of Lc from
the endosome to the cytosol. More surprising, bafilomycin also
inhibited functional internalization of recombinant BoNT/A Lc into
both neuroblastoma and non-neuronal cells in the absence of Hc,
most obviously in Neuro2a and HIT-T15 cells (FIG. 7). As with RGCN
above, these experiments are complicated by the bafilomycin
cytotoxicity, which leads to increased background of SNAP25
cleavage following addition of BoNT/A Lc. This is most apparent in
293 cells which appear particularly susceptible to bafilomycin
toxicity. Despite this background though, it is clear that
bafilomycin blocks the enhanced cleavage of SNAP25 elicited by
incubation of cells with BoNT/A Lc in the presence of DNA
transfection reagents. Bafilomycin had no inhibitory effect on the
efficiency of plasmid DNA transfection mediated by FuGene in
Neuro2a, M17 or HIT-T15 cells as assessed by fluorescent protein
expression from a transfected expression vector (data not shown).
These results suggest that the DNA transfection reagent-mediated
transduction of BoNT/A Lc requires endosome acidification, and thus
occurs via a similar mechanism as occurs during holotoxin
intoxication.
Commercial DNA Transfection Reagents Enhance Cellular Uptake of
Other BoNT Serotypes.
[0120] Previous experiments showed that commercial DNA transfection
reagents facilitate BoNT toxin or Lc internalization into a variety
of neuronal and non-neuronal cell lines. To test whether DNA
transfection reagent-mediated intoxication might be unique to type
A toxin, botulinum toxin type B and type E were also tested using
this delivery system. Cleavage of SNAP25 was used as the indicator
for BoNT/E toxin or Lc internalization while reduction of VAMP2 was
used to monitor BoNT/B toxin or Lc internalization. As shown in
FIG. 8, only trace amounts of the substrate proteins normally
became cleaved following exposure of M17 or Neuro2a neuroblastoma
cells to even high concentrations of the BoNT holotoxins or Lc
protease. In contrast, inclusion of FuGene-HD promoted substantial
cleavage of the appropriate BoNT substrates for both BoNT/B and
BoNT/E. Similar results were obtained when the holotoxins were
replaced by purified recombinant Lc proteases of both toxin
serotypes. These results show that enhanced internalization of BoNT
Lc proteases by DNA transfection reagents occurs for serotypes that
naturally use different surface receptors to enter cells and may
function for all serotypes.
Example 4
Discussion
[0121] Although BoNT intoxication is exquisitely efficient within
animals and cultured primary neurons, it has not proved so
efficient in established cell lines and this has inhibited research
activities and cell-based drug screening efforts. Intoxication
occurs following receptor-mediated internalization of BoNT and then
transposition of the toxin light chain (Lc) protease from the
endosome to the cytosol. The uptake and then chaperoning of the
protease to the cytosol is mediated by the BoNT heavy chain (Hc).
Because BoNT Hc binds to receptors found specifically on neuronal
cells, non-neuronal cells have proven to be insensitive to the
toxin. A variety of neuroblastoma cell lines are available and,
while detectable BoNT intoxication will often take place (usually
measured by SNARE protein cleavage), it is generally far less
efficient than in primary neurons. Here it was shown that inclusion
of commercial DNA transfection reagents during BoNT intoxication
can significantly improve intoxication efficiencies and make
possible efficient BoNT protease internalization into non-neuronal
cells even in the absence of the BoNT heavy chain.
[0122] While it has been possible to experimentally deliver BoNT Lc
to cell cytosol by DNA transfection methods or cell
permeabilization, these methods result in cells that are often
damaged with high and likely uneven levels of Lc protein that
arrives in the cytosol by processes different than occur during
intoxication. The reagents used here to deliver BoNT to cells are
not considered particularly toxic to cells. These reagents are
widely used for DNA transfection reagents and are either
lipid-based (Lipofectamine 2000), polycationic polymers (PEI) or of
undefined chemistry (FuGene). Lipid-mediated DNA transfection is
reported to occur via endocytosis and release from endosomes and
thus follows similar route into as botulinum toxin entry into
neurons. While lipid-based reagents have been used to deliver
proteins to cells in a process called transduction, the reagents
used in this study are not generally used for this purpose. Some
studies have identified other lipid-based reagents that are
effective for protein transduction although they have not been
studied for delivery of BoNT proteases and were not tested here. An
early report of intoxication after liposomal delivery of BoNT Lc to
motor neurons in animals was not apparently followed up in cultured
cells.
[0123] BoNT holotoxin intoxication of at least some neuroblastoma
cells can be substantially improved by the presence of certain DNA
transfection reagents such as FuGene-HD, Lipofectamine 2000 and
polyethylenimine (PEI). Furthermore, these reagents permit
intoxication of some non-neuronal cell types that are not normally
susceptible to BoNT intoxication. Finally, the DNA transfection
reagents facilitate intoxication of neuronal and non-neuronal cells
exposed only to isolated BoNT Lc proteases. Since the non-neuronal
cells are thought to lack the protein receptors recognized by BoNT,
and since the isolated BoNT Lcs lack the receptor binding domains,
the results imply that the DNA transfection reagents are
facilitating uptake of BoNT through a process independent of cell
surface protein receptors. Lipid-based DNA transfection reagents,
such as used in our studies, facilitate DNA endocytosis in a wide
array of cell types in a process not shown to involve protein
receptors. It is likely that these reagents perform a similar role
in BoNT transduction. It is believed that the DNA transfection
reagents facilitate the endocytosis of BoNT holotoxin or isolated
BoNT Lc, even in the absence of BoNT cell surface receptors.
[0124] Primary neurons are the most sensitive cells to BoNT
holotoxin intoxication, requiring as little as picomolar quantities
of some serotypes to produce effects on the cells. Use of DNA
transfection reagents did not improve the efficiency of BoNT/A
intoxication of primary rat cerebellar granule neurons (not shown).
Detectable DNA transfection in these cells were achieved with these
reagents. It is not known whether the inability to improve
intoxication efficiency was due to poor responsiveness to DNA
transfection reagents in primary cerebellar neurons, or because
receptor-mediated internalization is not limiting in these
cells.
[0125] Typically neuroblastoma cells are sensitive to a more
limited range of BoNT serotypes than primary neurons and require
higher BoNT concentrations to achieve measurable intoxication. The
neuroblastoma cells used in this study, Neuro2a and M17, required
nanomolar amounts of BoNT serotypes A, B and E for detectable
cleavage of their SNARE protein substrates. Neuro2a cells, which
are the least sensitive of the two lines, were found to become
several orders of magnitude more sensitive to these BoNT serotypes
in the presence of DNA transfection reagents. The BoNT sensitivity
of M17 cells was also improved substantially by these reagents.
BoNT/A intoxication of a third neuroblastoma cell line, PC12, was
only slightly improved by DNA transfection reagents (not shown).
DNA transfection in this cell line with these reagents was also
very poor and may explain the poor enhancement of intoxication. The
results indicate that, for neuroblastoma cells susceptible to DNA
transfection, it is possible to achieve BoNT intoxication with
sensitivities close to those obtained in primary neurons.
[0126] To further characterize DNA transfection reagent enhanced
intoxication, the time required to achieve intoxication in the
presence or absence of these reagents in Neuro2a cells were
compared. First it was shown that delivery of functional BoNT Lc to
the cell cytosol was both time and dose dependent. Some SNARE
protein cleavage could be detected as soon as 30 m following
addition of 10 nM BoNT/A in the presence of FuGene-HD, while almost
no cleavage could be detected after 24 hours in the absence of
FuGene. In a separate study, Neuro2a cells were exposed to toxin
for variable amounts of time with FuGene-HD, then washed and
cultured an additional 24 hours before being tested for SNAP25
cleavage. These results showed that exposure to BoNT/A for more
than 2 hours did not improve the level of SNAP25 cleavage detected
a day later (data not shown). Extending the culture time beyond a
day also did not improve the level of SNAP25 cleavage. These
results suggest that virtually all of the Neuro2a cells that are
susceptible to BoNT/A in FuGene-HD have endocytosed toxin by two
hours and that the small amount of SNAP25 that remains intact in
the population probably derives from a subset of cells that remain
refractory and have not internalized BoNT/A.
[0127] Consistent with the hypothesis that the DNA transfection
reagents facilitate receptor-independent uptake of BoNT, it was
shown that these reagents make it possible to achieve BoNT
intoxication of non-neuronal cells not normally susceptible to the
toxin. The two cell lines studied, HEK293 and HIT-T15 are both
commonly used cell lines for studies of protein secretion and both
contain SNARE protein substrates for BoNT. In both cell lines,
easily detected SNARE protein cleavage could be detected following
incubation with 10 nM BoNT/A in the presence of FuGene-HD or
Lipofectamine 2000. It is possible that the improved intoxication
efficacy obtained with lipid-based DNA transfection reagents is
aided by the enhanced SNARE protein cleavage activity that has been
reported for some BoNT serotypes in the presence of charged lipid
mixtures. This would require that the lipid components remain
associated with the BoNT Lc following uptake and translocation and
promote improved substrate cleavage in the cytosol. Whatever
mechanisms are involved, the ability to achieve BoNT intoxication
of various neuronal and non-neuronal secretory cell lines without
the need to transfect or permeabilize the cells should have useful
research applications.
[0128] It was tested whether enhanced BoNT holotoxin intoxication
of cells required endosomal acidification as was previously shown
to be necessary for natural intoxication. Bafilomycin inhibits
endosome acidification and inhibits natural intoxication,
presumably by altering the ability of the BoNT Hc to serve as a
chaperone for transcytosis of BoNT Lc. In contrast, DNA
transfection as mediated by lipid-based reagents does not require
acidification for function transfection of cells. BoNT intoxication
of both neuronal and non-neuronal cell lines, in the presence or
absence of DNA transfection reagents, was clearly inhibited by
bafilomycin. Thus, although the reagents appear to promote a
receptor-independent endocytosis of BoNT, the subsequent step in
the intoxication process, specifically translocation from the
endosome to the cytosol, appears to take place by natural
intoxication processes. This suggests that cells intoxicated by
BoNT in the presence of DNA transfection reagents remain good
models of naturally intoxicated cells.
[0129] Since the DNA transfection reagents obviate the need for
cell surface receptors during BoNT intoxication, it is
understandable that this could also obviate the need for the BoNT
Hc receptor binding domain to achieve internalization of BoNT Lc.
It is much more difficult to explain how the BoNT Lc is transferred
to the cytosol following endocytosis in the absence of the Hc
translocation domain, yet this clearly occurs. The results show
that isolated BoNT Lc for serotypes A, B and E are each capable of
efficient internalization to the cytosol and consequent SNARE
protein cleavage when applied some neuronal and non-neuronal cells
in the presence of DNA transfection reagents. The molar amount of
BoNT Lc required to produce SNARE protein cleavage was similar to
that required for holotoxin intoxication, indicating that the
intoxication efficiency is not significantly reduced when BoNT Lc
is delivered to cells with DNA transfection reagents in the absence
of Hc. Surprisingly, this functional transduction of BoNT Lc was
fully sensitive to bafilomycin indicating that endosome
acidification is a critical component of the BoNT Lc transduction
process. This result appears inconsistent with prior reports that
BoNT Hc is required for Lc translocation from the endosome to the
cytosol. It is believed that the DNA transfection reagents may
remain associated with the BoNT Lc and provides the chaperone
function normally performed by the Hc. Methods permitting BoNT Lc
internalization through the endosome in the absence of He should
permit experiments to further elucidate the pathways and mechanisms
of BoNT Lc translocation and intracellular transport.
[0130] The ability to intoxicate cultured neuronal and non-neuronal
cells by a process that mimics that of native BoNT without the need
for holotoxin reduces risks to workers and simplifies the facility
requirements. This could be particularly useful in the performance
of high throughput screening for BoNT inhibitors using cell-based
assays.
[0131] The teachings of U.S. patent application Ser. No.
12/481,889, filed Jun. 10, 2009, entitled "Designer Ubiquitin
Ligases for Regulation of Intracellular Pathogenic Proteins" by
Shoemaker, Charles, et al. are incorporated herein by reference in
its entirety.
[0132] The teachings of PCT Application No. PCT/US10/21479, filed
Jan. 20, 2010, entitled "Methods For The Delivery Of Toxins Or
Enzymatically Active Portions Thereof" by George A. Oyler, et al.
are incorporated herein by reference in its entirety.
[0133] The relevant teachings of all the references, patents and/or
patent applications cited herein are incorporated herein by
reference in their entirety.
[0134] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
2211323DNAClostridium botulinum 1atgccgtttg tgaacaaaca gtttaactat
aaagatccgg tgaacggcgt ggatattgcg 60tatattaaaa ttccgaacgt gggccagatg
cagccggtga aagcgtttaa aattcataac 120aaaatttggg tgattccgga
acgcgatacc tttaccaacc cggaagaagg cgatctgaac 180ccgccgccgg
aagcgaaaca ggtgccggtg agctattatg atagcaccta tctgagcacc
240gataacgaaa aagataacta tctgaaaggc gtgaccaaac tgtttgaacg
catttatagc 300accgatctgg gccgcatgct gctgaccagc attgtgcgcg
gcattccgtt ttggggcggc 360agcaccattg ataccgaact gaaagtgatt
gataccaact gcattaacgt gattcagccg 420gatggcagct atcgcagcga
agaactgaac ctggtgatta ttggcccgag cgcggatatt 480attcagtttg
aatgcaaaag ctttggccat gaagtgctga acctgacccg caacggctat
540ggcagcaccc agtatattcg ctttagcccg gattttacct ttggctttga
agaaagcctg 600gaagtggata ccaacccgct gctgggcgcg ggcaaatttg
cgaccgatcc ggcggtgacc 660ctggcgcatg aactgattca tgcgggccat
cgcctgtatg gcattgcgat taacccgaac 720cgcgtgttta aagtgaacac
caacgcgtat tatgaaatga gcggcctgga agtgagcttt 780gaagaactgc
gcacctttgg cggccatgat gcgaaattta ttgatagcct gcaggaaaac
840gaatttcgcc tgtattatta taacaaattt aaagatattg cgagcaccct
gaacaaagcg 900aaaagcattg tgggcaccac cgcgagcctg cagtatatga
aaaacgtgtt taaagaaaaa 960tatctgctga gcgaagatac cagcggcaaa
tttagcgtgg ataaactgaa atttgataaa 1020ctgtataaaa tgctgaccga
aatttatacc gaagataact ttgtgaaatt ttttaaagtg 1080ctgaaccgca
aaacctatct gaactttgat aaagcggtgt ttaaaattaa cattgtgccg
1140aaagtgaact ataccattta tgatggcttt aacctgcgca acaccaacct
ggcggcgaac 1200tttaacggcc agaacaccga aattaacaac atgaacttta
ccaaactgaa aaactttacc 1260ggcctgtttg aattttataa actgctgtgc
gtgcgcggca ttattaccag caaaaccaaa 1320tag 13232440PRTClostridium
botulinum 2Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val
Asn Gly 1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val 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 Thr 65 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 Ile 145 150 155 160 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 Asn 225 230 235
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 Lys 305 310 315 320 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 Asn 385 390 395 400 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 435 440 31347DNAClostridium botulinum 3atgccgtttg tgaacaaaca
gtttaactat aaagatccgg tgaacggcgt ggatattgcg 60tatattaaaa ttccgaacgc
gggccagatg cagccggtga aagcgtttaa aattcataac 120aaaatttggg
tgattccgga acgcgatacc tttaccaacc cggaagaagg cgatctgaac
180ccgccgccgg aagcgaaaca ggtgccggtg agctattatg atagcaccta
tctgagcacc 240gataacgaaa aagataacta tctgaaaggc gtgaccaaac
tgtttgaacg catttatagc 300accgatctgg gccgcatgct gctgaccagc
attgtgcgcg gcattccgtt ttggggcggc 360agcaccattg ataccgaact
gaaagtgatt gataccaact gcattaacgt gattcagccg 420gatggcagct
atcgcagcga agaactgaac ctggtgatta ttggcccgag cgcggatatt
480attcagtttg aatgcaaaag ctttggccat gatgtgctga acctgacccg
caacggctat 540ggcagcaccc agtatattcg ctttagcccg gattttacct
ttggctttga agaaagcctg 600gaagtggata ccaacccgct gctgggcgcg
ggcaaatttg cgaccgatcc ggcggtgacc 660ctggcgcatg aactgattca
tgcggaacat cgcctgtatg gcattgcgat taacccgaac 720cgcgtgttta
aagtgaacac caacgcgtat tatgaaatga gcggcctgga agtgagcttt
780gaagaactgc gcacctttgg cggccatgat gcgaaattta ttgatagcct
gcaggaaaac 840gaatttcgcc tgtattatta taacaaattt aaagatgtgg
cgagcaccct gaacaaagcg 900aaaagcatta ttggcaccac cgcgagcctg
cagtatatga aaaacgtgtt taaagaaaaa 960tatctgctga gcgaagatac
cagcggcaaa tttagcgtgg ataaactgaa atttgataaa 1020ctgtataaaa
tgctgaccga aatttatacc gaagataact ttgtgaactt ttttaaagtg
1080attaaccgca aaacctatct gaactttgat aaagcggtgt ttcgcattaa
cattgtgccg 1140gatgaaaact ataccattaa agatggcttt aacctgaaag
gcgcgaacct gagcaccaac 1200tttaacggcc agaacaccga aattaacagc
cgcaacttta cccgcctgaa aaactttacc 1260ggcctgtttg aattttataa
actgctgtgc gtgcgcggca ttattccgtt taaaaccaaa 1320agcctggatg
aaggctataa caaatag 13474448PRTClostridium botulinum 4Met Pro Phe
Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly 1 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 Thr 65 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 Ile 145 150 155
160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Asp 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 Glu His Arg Leu
Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240 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 Val Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Ile
290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys
Glu Lys 305 310 315 320 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 Asn Phe Phe Lys
Val Ile Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val
Phe Arg Ile Asn Ile Val Pro Asp Glu Asn Tyr 370 375 380 Thr Ile Lys
Asp Gly Phe Asn Leu Lys Gly Ala Asn Leu Ser Thr Asn 385 390 395 400
Phe Asn Gly Gln Asn Thr Glu Ile Asn Ser Arg Asn Phe Thr Arg 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 Pro Phe Lys Thr Lys Ser Leu Asp Glu Gly
Tyr Asn Lys 435 440 445 51335DNAClostridium botulinum 5atgccgtttg
tgaacaaacc gtttaactat cgcgatccgg gcaacggcgt ggatattgcg 60tatattaaaa
ttccgaacgc gggccagatg cagccggtga aagcgtttaa aattcatgaa
120ggcgtgtggg tgattccgga acgcgatacc tttaccaacc cggaagaagg
cgatctgaac 180ccgccgccgg aagcgaaaca ggtgccggtg agctattatg
atagcaccta tctgagcacc 240gataacgaaa aagataacta tctgaaaggc
gtgattaaac tgtttgatcg catttatagc 300accggcctgg gccgcatgct
gctgagcttt attgtgaaag gcattccgtt ttggggcggc 360agcaccattg
ataccgaact gaaagtgatt gataccaact gcattaacgt gattgaaccg
420ggcggcagct atcgcagcga agaactgaac ctggtgatta ccggcccgag
cgcggatatt 480attcagtttg aatgcaaaag ctttggccat gatgtgttta
acctgacccg caacggctat 540ggcagcaccc agtatattcg ctttagcccg
gattttacct ttggctttga agaaagcctg 600gaagtggata ccaacccgct
gctgggcgcg ggcacctttg cgaccgatcc ggcggtgacc 660ctggcgcatg
aactgattca tgcggcgcat cgcctgtatg gcattgcgat taacccgaac
720cgcgtgctga aagtgaaaac caacgcgtat tatgaaatga gcggcctgga
agtgagcttt 780gaagaactgc gcacctttgg cggcaacgat accaacttta
ttgatagcct gtggcagaaa 840aaatttagcc gcgatgcgta tgataacctg
cagaacattg cgcgcattct gaacgaagcg 900aaaaccattg tgggcaccac
caccccgctg cagtatatga aaaacatttt tattcgcaaa 960tattttctga
gcgaagatgc gagcggcaaa attagcgtga acaaagcggc gtttaaagaa
1020ttttatcgcg tgctgacccg cggctttacc gaactggaat ttgtgaaccc
gtttaaagtg 1080attaaccgca aaacctatct gaactttgat aaagcggtgt
ttcgcattaa cattgtgccg 1140gatgaaaact ataccattaa cgaaggcttt
aacctggaag gcgcgaacag caacggccag 1200aacaccgaaa ttaacagccg
caactttacc cgcctgaaaa actttaccgg cctgtttgaa 1260ttttataaac
tgctgtgcgt gcgcggcatt attccgttta aaaccaaaag cctggatgaa
1320ggctataaca aatag 13356444PRTClostridium botulinum 6Met Pro Phe
Val Asn Lys Pro Phe Asn Tyr Arg Asp Pro Gly Asn Gly 1 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 Glu Gly Val 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 Thr 65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly
Val Ile Lys Leu Phe Asp 85 90 95 Arg Ile Tyr Ser Thr Gly Leu Gly
Arg Met Leu Leu Ser Phe Ile Val 100 105 110 Lys 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 Glu Pro Gly Gly Ser Tyr 130 135 140 Arg Ser
Glu Glu Leu Asn Leu Val Ile Thr Gly Pro Ser Ala Asp Ile 145 150 155
160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Asp Val Phe 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 Thr Phe Ala Thr Asp Pro Ala
Val Thr Leu Ala His Glu 210 215 220 Leu Ile His Ala Ala His Arg Leu
Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240 Arg Val Leu Lys Val
Lys 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 Asn Asp Thr Asn 260 265 270 Phe
Ile Asp Ser Leu Trp Gln Lys Lys Phe Ser Arg Asp Ala Tyr Asp 275 280
285 Asn Leu Gln Asn Ile Ala Arg Ile Leu Asn Glu Ala Lys Thr Ile Val
290 295 300 Gly Thr Thr Thr Pro Leu Gln Tyr Met Lys Asn Ile Phe Ile
Arg Lys 305 310 315 320 Tyr Phe Leu Ser Glu Asp Ala Ser Gly Lys Ile
Ser Val Asn Lys Ala 325 330 335 Ala Phe Lys Glu Phe Tyr Arg Val Leu
Thr Arg Gly Phe Thr Glu Leu 340 345 350 Glu Phe Val Asn Pro Phe Lys
Val Ile Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val
Phe Arg Ile Asn Ile Val Pro Asp Glu Asn Tyr 370 375 380 Thr Ile Asn
Glu Gly Phe Asn Leu Glu Gly Ala Asn Ser Asn Gly Gln 385 390 395 400
Asn Thr Glu Ile Asn Ser Arg Asn Phe Thr Arg Leu Lys Asn Phe Thr 405
410 415 Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg Gly Ile Ile
Pro 420 425 430 Phe Lys Thr Lys Ser Leu Asp Glu Gly Tyr Asn Lys 435
440 71326DNAClostridium botulinum 7atgccggtga ccattaacaa ctttaactat
aacgatccga ttgataacaa caacattatt 60atgatggaac cgccgtttgc gcgcggcacc
ggccgctatt ataaagcgtt taaaattacc 120gatcgcattt ggattattcc
ggaacgctat acctttggct ataaaccgga agattttaac 180aaaagcagcg
gcatttttaa ccgcgatgtg tgcgaatatt atgatccgga ttatctgaac
240accaacgata aaaaaaacat ttttctgcag accatgatta aactgtttaa
ccgcattaaa 300agcaaaccgc tgggcgaaaa actgctggaa atgattatta
acggcattcc gtatctgggc 360gatcgccgcg tgccgctgga agaatttaac
accaacattg cgagcgtgac cgtgaacaaa 420ctgattagca acccgggcga
agtggaacgc aaaaaaggca tttttgcgaa cctgattatt 480tttggcccgg
gcccggtgct gaacgaaaac gaaaccattg atattggcat tcagaaccat
540tttgcgagcc gcgaaggctt tggcggcatt atgcagatga aattttgccc
ggaatatgtg 600agcgtgttta acaacgtgca ggaaaacaaa ggcgcgagca
tttttaaccg ccgcggctat 660tttagcgatc cggcgctgat tctgatgcat
gaactgattc atgtgctgca tggcctgtat 720ggcattaaag tggatgatct
gccgattgtg ccgaacgaaa aaaaattttt tatgcagagc 780accgatgcga
ttcaggcgga agaactgtat acctttggcg gccaggatcc gagcattatt
840accccgagca ccgataaaag catttatgat aaagtgctgc agaactttcg
cggcattgtg 900gatcgcctga acaaagtgct ggtgtgcatt agcgatccga
acattaacat taacatttat 960aaaaacaaat ttaaagataa atataaattt
gtggaagata gcgaaggcaa atatagcatt 1020gatgtggaaa gctttgataa
actgtataaa agcctgatgt ttggctttac cgaaaccaac 1080attgcggaaa
actataaaat taaaacccgc gcgagctatt ttagcgatag cctgccgccg
1140gtgaaaatta aaaacctgct ggataacgaa atttatacca ttgaagaagg
ctttaacatt 1200agcgataaag atatggaaaa agaatatcgc ggccagaaca
aagcgattaa caaacaggcg 1260tatgaagaaa ttagcaaaga acatctggcg
gtgtataaaa ttcagatgtg caaaagcgtg 1320aaatag 13268441PRTClostridium
botulinum 8Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile
Asp Asn 1 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 Asn 65 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 Ile 145 150 155 160 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
Tyr 225 230 235 240 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 Tyr 305 310 315 320 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 Ile 385 390 395 400 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 435 440 91323DNAClostridium botulinum
9atgccggtga ccattaacaa ctttaactat aacgatccga ttgataacga taacattatt
60atgatggaac cgccgtttgc gcgcggcacc ggccgctatt ataaagcgtt taaaattacc
120gatcgcattt ggattattcc ggaacgctat acctttggct ataaaccgga
agattttaac 180aaaagcagcg gcatttttaa ccgcgatgtg tgcgaatatt
atgatccgga ttatctgaac 240accaacgata aaaaaaacat ttttctgcag
accatgatta aactgtttaa ccgcattaaa 300agcaaaccgc tgggcgaaaa
actgctggaa atgattatta acggcattcc gtatctgggc 360gatcgccgcg
tgccgctgga agaatttaac accaacattg cgagcgtgac cgtgaacaaa
420ctgattagca acccgggcga agtggaacag aaaaaaggca tttttgcgaa
cctgattatt 480tttggcccgg gcccggtgct gaacgaaaac gaaaccattg
atattggcat tcagaaccat 540tttgcgagcc gcgaaggctt tggcggcatt
atgcagatga aattttgccc ggaatatgtg 600agcgtgttta acaacgtgca
ggaaaacaaa ggcgcgagca tttttaaccg ccgcggctat 660tttagcgatc
cggcgctgat tctgatgcat gaactgattc atgtgctgca tggcctgtat
720ggcattaaag tggatgatct gccgattgtg ccgaacgaaa aaaaattttt
tatgcagagc 780accgatacca ttcaggcgga agaactgtat acctttggcg
gccaggatcc gagcattatt 840agcccgagca ccgataaaag catttatgat
aaagtgctgc agaactttcg cggcattgtg 900gatcgcctga acaaagtgct
ggtgtgcatt agcgatccga acattaacat taacatttat 960aaaaacaaat
ttaaagataa atataaattt gtggaagata gcgaaggcaa atatagcatt
1020gatgtggaaa gctttaacaa actgtataaa agcctgatgt ttggctttac
cgaaattaac 1080attgcggaaa actataaaat taaaacccgc gcgagctatt
ttagcgatag cctgccgccg 1140gtgaaaatta aaaacctgct ggataacgaa
atttatacca ttgaagaagg ctttaacatt 1200agcgataaaa acatgggcaa
agaatatcgc ggccagaaca aagcgattaa caaacaggcg 1260tatgaagaaa
ttagcaaaga acatctggcg gtgtataaaa ttcagatgtg caaaagcgtg 1320tag
132310440PRTClostridium botulinum 10Met Pro Val Thr Ile Asn Asn Phe
Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 Asp 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 Asn 65
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 Gln
Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 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 Tyr 225 230 235 240 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 Thr
Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro
Ser Ile Ile Ser 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 Tyr 305 310
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 Asn Lys Leu Tyr
Lys Ser Leu 340 345 350 Met Phe Gly Phe Thr Glu Ile 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 Ile 385 390 395 400 Ser Asp Lys Asn
Met Gly 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 435 440 111350DNAClostridium
botulinum 11atgccgatta ccattaacaa ctttaactat agcgatccgg tggataacaa
aaacattctg 60tatctggata cccatctgaa caccctggcg aacgaaccgg aaaaagcgtt
tcgcattacc 120ggcaacattt gggtgattcc ggatcgcttt agccgcaaca
gcaacccgaa cctgaacaaa 180ccgccgcgcg tgaccagccc gaaaagcggc
tattatgatc cgaactatct gagcaccgat 240agcgataaag atccgtttct
gaaagaaatt attaaactgt ttaaacgcat taacagccgc 300gaaattggcg
aagaactgat ttatcgcctg agcaccgata ttccgtttcc gggcaacaac
360aacaccccga ttaacacctt tgattttgat gtggatttta acagcgtgga
tgtgaaaacc 420cgccagggca acaactgggt gaaaaccggc agcattaacc
cgagcgtgat tattaccggc 480ccgcgcgaaa acattattga tccggaaacc
agcaccttta aactgaccaa caacaccttt 540gcggcgcagg aaggctttgg
cgcgctgagc attattagca ttagcccgcg ctttatgctg 600acctatagca
acgcgaccaa cgatgtgggc gaaggccgct ttagcaaaag cgaattttgc
660atggatccga ttctgattct gatgcatgaa ctgaaccatg cgatgcataa
cctgtatggc 720attgcgattc cgaacgatca gaccattagc agcgtgacca
gcaacatttt ttatagccag 780tataacgtga aactggaata tgcggaaatt
tatgcgtttg gcggcccgac cattgatctg 840attccgaaaa gcgcgcgcaa
atattttgaa gaaaaagcgc tggattatta tcgcagcatt 900gcgaaacgcc
tgaacagcat taccaccgcg aacccgagca gctttaacaa atatattggc
960gaatataaac agaaactgat tcgcaaatat cgctttgtgg tggaaagcag
cggcgaagtg 1020accgtgaacc gcaacaaatt tgtggaactg tataacgaac
tgacccagat ttttaccgaa 1080tttaactatg cgaaaattta taacgtgcag
aaccgcaaaa tttatctgag caacgtgtat 1140accccggtga ccgcgaacat
tctggatgat aacgtgtatg atattcagaa cggctttaac 1200attccgaaaa
gcaacctgaa cgtgctgttt atgggccaga acctgagccg caacccggcg
1260ctgcgcaaag tgaacccgga aaacatgctg tatctgttta ccaaattttg
ccataaagcg 1320attgatggcc gcagcctgta taacaaatag
135012449PRTClostridium botulinum 12Met Pro Ile Thr Ile Asn Asn Phe
Asn Tyr Ser Asp Pro Val Asp Asn 1 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 Asp 65
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 Gly 145 150 155 160 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 Gly 225 230 235 240 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 Gly 305 310
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 Asn 385 390 395 400 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 131329DNAClostridium botulinum 13atgacctggc cggtgaaaga
ttttaactat agcgatccgg tgaacgataa cgatattctg 60tatctgcgca ttccgcagaa
caaactgatt accaccccgg tgaaagcgtt tatgattacc 120cagaacattt
gggtgattcc ggaacgcttt agcagcgata ccaacccgag cctgagcaaa
180ccgccgcgcc cgaccagcaa atatcagagc tattatgatc cgagctatct
gagcaccgat 240gaacagaaag atacctttct gaaaggcatt attaaactgt
ttaaacgcat taacgaacgc 300gatattggca aaaaactgat taactatctg
gtggtgggca gcccgtttat gggcgatagc 360agcaccccgg aagatacctt
tgattttacc cgccatacca ccaacattgc ggtggaaaaa 420tttgaaaacg
gcagctggaa agtgaccaac attattaccc cgagcgtgct gatttttggc
480ccgctgccga acattctgga ttataccgcg agcctgaccc tgcagggcca
gcagagcaac 540ccgagctttg aaggctttgg caccctgagc attctgaaag
tggcgccgga atttctgctg 600acctttagcg atgtgaccag caaccagagc
agcgcggtgc tgggcaaaag cattttttgc 660atggatccgg tgattgcgct
gatgcatgaa ctgacccata gcctgcatca gctgtatggc 720attaacattc
cgagcgataa acgcattcgc ccgcaggtga gcgaaggctt ttttagccag
780gatggcccga acgtgcagtt tgaagaactg tatacctttg gcggcctgga
tgtggaaatt 840attccgcaga ttgaacgcag ccagctgcgc gaaaaagcgc
tgggccatta taaagatatt 900gcgaaacgcc tgaacaacat taacaaaacc
attccgagca gctggattag caacattgat 960aaatataaaa aaatttttag
cgaaaaatat aactttgata aagataacac cggcaacttt 1020gtggtgaaca
ttgataaatt taacagcctg tatagcgatc tgaccaacgt gatgagcgaa
1080gtggtgtata gcagccagta taacgtgaaa aaccgcaccc attattttag
ccgccattat 1140ctgccggtgt ttgcgaacat tctggatgat aacatttata
ccattcgcga tggctttaac 1200ctgaccaaca aaggctttaa cattgaaaac
agcggccaga acattgaacg caacccggcg 1260ctgcagaaac tgagcagcga
aagcgtggtg gatctgttta ccaaagtgtg cctgcgcctg 1320accaaatag
132914442PRTClostridium botulinum 14Met Thr Trp Pro Val Lys Asp Phe
Asn Tyr Ser Asp Pro Val Asn Asp 1 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 Asp 65
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 Gly 145 150 155 160 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 Gly 225 230 235 240 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 Asp 305 310
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 Asn 385 390 395 400 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 435 440 151269DNAClostridium botulinum 15atgccgaaaa
ttaacagctt taactataac gatccggtga acgatcgcac cattctgtat 60attaaaccgg
gcggctgcca ggaattttat aaaagcttta acattatgaa aaacatttgg
120attattccgg aacgcaacgt gattggcacc accccgcagg attttcatcc
gccgaccagc 180ctgaaaaacg gcgatagcag ctattatgat ccgaactatc
tgcagagcga tgaagaaaaa 240gatcgctttc tgaaaattgt gaccaaaatt
tttaaccgca ttaacaacaa cctgagcggc 300ggcattctgc tggaagaact
gagcaaagcg aacccgtatc tgggcaacga taacaccccg 360gataaccagt
ttcatattgg cgatgcgagc gcggtggaaa ttaaatttag caacggcagc
420caggatattc tgctgccgaa cgtgattatt atgggcgcgg aaccggatct
gtttgaaacc 480aacagcagca acattagcct gcgcaacaac tatatgccga
gcaaccatcg ctttggcagc 540attgcgattg tgacctttag cccggaatat
agctttcgct ttaacgataa ctgcatgaac 600gaatttattc aggatccggc
gctgaccctg atgcatgaac tgattcatag cctgcatggc 660ctgtatggcg
cgaaaggcat taccaccaaa tataccatta cccagaaaca gaacccgctg
720attaccaaca ttcgcggcac caacattgaa gaatttctga cctttggcgg
caccgatctg 780aacattatta ccagcgcgca gagcaacgat atttatacca
acctgctggc ggattataaa 840aaaattgcga gcaaactgag caaagtgcag
gtgagcaacc cgctgctgaa cccgtataaa 900gatgtgtttg aagcgaaata
tggcctggat aaagatgcga gcggcattta tagcgtgaac 960attaacaaat
ttaacgatat ttttaaaaaa ctgtatagct ttaccgaatt tgatctggcg
1020accaaatttc aggtgaaatg ccgccagacc tatattggcc agtataaata
ttttaaactg 1080agcaacctgc tgaacgatag catttataac attagcgaag
gctataacat taacaacctg 1140aaagtgaact ttcgcggcca gaacgcgaac
ctgaacccgc gcattattac cccgattacc 1200ggccgcggcc tggtgaaaaa
aattattcgc ttttgcaaaa acattgtgag cgtgaaaggc 1260attcgctag
126916422PRTClostridium botulinum 16Met Pro Lys Ile Asn Ser Phe Asn
Tyr Asn Asp Pro Val Asn Asp Arg 1 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 Lys 65
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 Thr 145 150 155 160 Asn Ser Ser
Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Arg
Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185
190 Arg Phe Asn Asp Asn Cys 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 Leu 225 230 235 240 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 Asn 305 310
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 Thr 385 390 395 400 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 420 171311DNAClostridium botulinum 17atgccggtgg
cgattaacag ctttaactat aacgatccgg tgaacgatga taccattctg 60tatatgcaga
ttccgtatga agaaaaaagc aaaaaatatt ataaagcgtt tgaaattatg
120cgcaacgtgt ggattattcc ggaacgcaac accattggca ccaacccgag
cgattttgat 180ccgccggcga gcctgaaaaa cggcagcagc gcgtattatg
atccgaacta tctgaccacc 240gatgcggaaa aagatcgcta tctgaaaacc
accattaaac tgtttaaacg cattaacagc 300aacccggcgg gcaaagtgct
gctgcaggaa attagctatg cgaaaccgta tctgggcaac 360gatcataccc
cgattgatga atttagcccg gtgacccgca ccaccagcgt gaacattaaa
420ctgagcacca acgtggaaag cagcatgctg ctgaacctgc tggtgctggg
cgcgggcccg 480gatatttttg aaagctgctg ctatccggtg cgcaaactga
ttgatccgga tgtggtgtat 540gatccgagca actatggctt tggcagcatt
aacattgtga cctttagccc ggaatatgaa 600tataccttta acgatattag
cggcggccat aacagcagca ccgaaagctt tattgcggat 660ccggcgatta
gcctggcgca tgaactgatt catgcgctgc atggcctgta tggcgcgcgc
720ggcgtgacct atgaagaaac cattgaagtg aaacaggcgc cgctgatgat
tgcggaaaaa 780ccgattcgcc tggaagaatt tctgaccttt ggcggccagg
atctgaacat tattaccagc 840gcgatgaaag aaaaaattta taacaacctg
ctggcgaact atgaaaaaat tgcgacccgc 900ctgagcgaag tgaacagcgc
gccgccggaa tatgatatta acgaatataa agattatttt 960cagtggaaat
atggcctgga taaaaacgcg gatggcagct ataccgtgaa cgaaaacaaa
1020tttaacgaaa tttataaaaa actgtatagc tttaccgaaa gcgatctggc
gaacaaattt 1080aaagtgaaat gccgcaacac ctattttatt aaatatgaat
ttctgaaagt gccgaacctg 1140ctggatgatg atatttatac cgtgagcgaa
ggctttaaca ttggcaacct ggcggtgaac 1200aaccgcggcc agagcattaa
actgaacccg aaaattattg atagcattcc ggataaaggc 1260ctggtggaaa
aaattgtgaa attttgcaaa agcgtgattc cgcgcaaata g
131118436PRTClostridium botulinum 18Met Pro Val Ala Ile Asn Ser Phe
Asn Tyr Asn Asp Pro Val Asn Asp 1 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 Thr 65
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 Pro 145 150 155 160 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 Arg 225 230 235 240 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 Phe 305 310
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 Asn 385 390 395 400 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 435 191314DNAClostridium barati 19atgccggtga
acattaacaa ctttaactat aacgatccga ttaacaacac caccattctg 60tatatgaaaa
tgccgtatta tgaagatagc aacaaatatt ataaagcgtt tgaaattatg
120gataacgtgt ggattattcc ggaacgcaac attattggca aaaaaccgag
cgatttttat 180ccgccgatta gcctggatag cggcagcagc gcgtattatg
atccgaacta tctgaccacc 240gatgcggaaa aagatcgctt tctgaaaacc
gtgattaaac tgtttaaccg cattaacagc 300aacccggcgg gccaggtgct
gctggaagaa attaaaaacg gcaaaccgta tctgggcaac 360gatcataccg
cggtgaacga attttgcgcg aacaaccgca gcaccagcgt ggaaattaaa
420gaaagcaacg gcaccaccga tagcatgctg ctgaacctgg tgattctggg
cccgggcccg 480aacattctgg aatgcagcac ctttccggtg cgcatttttc
cgaacaacat tgcgtatgat 540ccgagcgaaa aaggctttgg cagcattcag
ctgatgagct ttagcaccga atatgaatat 600gcgtttaacg ataacaccga
tctgtttatt gcggatccgg cgattagcct ggcgcatgaa 660ctgattcatg
tgctgcatgg cctgtatggc gcgaaaggcg tgaccaacaa aaaagtgatt
720gaagtggatc agggcgcgct gatggcggcg gaaaaagata ttaaaattga
agaatttatt 780acctttggcg gccaggatct gaacattatt accaacagca
ccaaccagaa aatttatgtg 840attctgctga gcaactatac cgcgattgcg
agccgcctga gccaggtgaa ccgcaacaac 900agcgcgctga acaccaccta
ttataaaaac ttttttcagt ggaaatatgg cctggatcag 960gatagcaacg
gcaactatac cgtgaacatt agcaaattta acgcgattta taaaaaactg
1020tttagcttta ccgaatgcga tctggcgcag aaatttcagg tgaaaaaccg
cagcaactat 1080ctgtttcatt ttaaaccgtt tcgcctgctg gatctgctgg
atgataacat ttatagcatt 1140agcgaaggct ttaacattgg cagcctgcgc
gtgaacaaca acggccagaa cattaacctg 1200aacagccgca ttgtgggccc
gattccggat aacggcctgg tggaacgctt tgtgggcctg 1260tgcaaaagca
ttgtgagcaa aaaaggcacc aaaaacagcc tgtgcattaa atag
131420437PRTClostridium barati 20Met Pro Val Asn Ile Asn Asn Phe
Asn Tyr Asn Asp Pro Ile Asn Asn 1 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 Thr 65
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 Pro 145 150 155 160 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 Ile 225 230 235 240 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 Gln 305 310
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 Leu 385 390 395 400 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 435 211311DNAClostridium botulinum 21atgccggtga
acattaaatt taactataac gatccgatta acaacgatga tattattatg 60atggaaccgt
ttaacgatcc gggcccgggc acctattata aagcgtttcg cattattgat
120cgcatttgga ttgtgccgga acgctttacc tatggctttc agccggatca
gtttaacgcg 180agcaccggcg tgtttagcaa agatgtgtat gaatattatg
atccgaccta tctgaaaacc 240gatgcggaaa aagataaatt tctgaaaacc
atgattaaac tgtttaaccg cattaacagc 300aaaccgagcg gccagcgcct
gctggatatg attgtggatg cgattccgta tctgggcaac 360gcgagcaccc
cgccggataa atttgcggcg aacgtggcga acgtgagcat taacaaaaaa
420attattcagc cgggcgcgga agatcagatt aaaggcctga tgaccaacct
gattattttt 480ggcccgggcc cggtgctgag cgataacttt accgatagca
tgattatgaa cggccatagc 540ccgattagcg aaggctttgg cgcgcgcatg
atgattcgct tttgcccgag ctgcctgaac 600gtgtttaaca acgtgcagga
aaacaaagat accagcattt ttagccgccg cgcgtatttt 660gcggatccgg
cgctgaccct gatgcatgaa ctgattcatg tgctgcatgg cctgtatggc
720attaaaatta gcaacctgcc gattaccccg aacaccaaag aattttttat
gcagcatagc 780gatccggtgc aggcggaaga actgtatacc tttggcggcc
atgatccgag cgtgattagc 840ccgagcaccg atatgaacat ttataacaaa
gcgctgcaga actttcagga tattgcgaac 900cgcctgaaca ttgtgagcag
cgcgcagggc agcggcattg atattagcct gtataaacag 960atttataaaa
acaaatatga ttttgtggaa gatccgaacg gcaaatatag cgtggataaa
1020gataaatttg ataaactgta taaagcgctg atgtttggct ttaccgaaac
caacctggcg 1080ggcgaatatg gcattaaaac ccgctatagc tattttagcg
aatatctgcc gccgattaaa 1140accgaaaaac tgctggataa caccatttat
acccagaacg aaggctttaa cattgcgagc 1200aaaaacctga aaaccgaatt
taacggccag aacaaagcgg tgaacaaaga agcgtatgaa 1260gaaattagcc
tggaacatct ggtgatttat cgcattgcga tgtgcaaata g
131122437PRTClostridium botulinumVARIANT(1)..(437)Xaa=Any Amino
Acid 22Met Pro Val Asn Ile Lys Xaa Phe Asn Tyr Asn Asp Pro Ile Asn
Asn 1 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 Lys 65 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 Ile 145 150 155 160 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 Tyr 225 230 235
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 Lys 305 310 315 320 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 Ala 385 390 395 400 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 435
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