U.S. patent application number 12/019188 was filed with the patent office on 2008-07-17 for gfp-snap25 fluorescence release assay for botulinum toxin protease activity.
This patent application is currently assigned to Allergan, Inc.. Invention is credited to Kei R. Aoki, Marcella A. Gilmore, Lance E. Steward.
Application Number | 20080171348 12/019188 |
Document ID | / |
Family ID | 35908131 |
Filed Date | 2008-07-17 |
United States Patent
Application |
20080171348 |
Kind Code |
A1 |
Steward; Lance E. ; et
al. |
July 17, 2008 |
GFP-SNAP25 Fluorescence Release Assay for Botulinum Toxin Protease
Activity
Abstract
The present invention provides a nucleic acid molecule which
contains a nucleotide sequence encoding a SNAP-25 substrate which
includes (i) a green fluorescent protein; (ii) a first partner of
an affinity couple; and (iii) a portion of SNAP-25 that includes a
BoNT/A, BoNT/C1 or BoNT/E recognition sequence containing a
cleavage site, where the cleavage site intervenes between the green
fluorescent protein and the first partner of the affinity couple.
Further provided herein is a nucleic acid molecule which contains a
nucleotide sequence encoding a tagged toxin substrate which
includes (i) a fluorescent protein; (ii) a first partner of an
affinity couple; and (iii) a clostridial toxin recognition sequence
containing a cleavage site, where the cleavage site intervenes
between the fluorescent protein and the first partner of the
affinity couple.
Inventors: |
Steward; Lance E.; (Irvine,
CA) ; Gilmore; Marcella A.; (Santa Ana, CA) ;
Aoki; Kei R.; (Coto de Caza, CA) |
Correspondence
Address: |
ALLERGEN, INC. (T2-7H)
2525 Dupont Drive
Irvine
CA
92612
US
|
Assignee: |
Allergan, Inc.
|
Family ID: |
35908131 |
Appl. No.: |
12/019188 |
Filed: |
January 24, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10917844 |
Aug 13, 2004 |
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12019188 |
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09942098 |
Aug 28, 2001 |
7332567 |
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10917844 |
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Current U.S.
Class: |
435/18 |
Current CPC
Class: |
G01N 33/56911 20130101;
C07K 14/001 20130101; C12Q 1/37 20130101; C07K 2319/00 20130101;
C12N 9/52 20130101; C07K 14/435 20130101; G01N 2333/33 20130101;
C12Y 304/24069 20130101; C07K 14/43595 20130101; C07K 14/705
20130101 |
Class at
Publication: |
435/18 |
International
Class: |
C12Q 1/37 20060101
C12Q001/37 |
Claims
1. A method of determining Botulinum toxin serotype E protease
activity, comprising the steps of: (a) treating with a sample, in
solution phase under conditions suitable for Botulinum toxin
serotype E protease activity, a tagged toxin substrate comprising
(i) a fluorescent protein; (ii) a first partner of an affinity
couple; and (iii) a Botulinum toxin serotype E recognition sequence
comprising a cleavage site, where the cleavage site intervenes
between said fluorescent protein and said first partner of the
affinity couple, such that a fluorescent cleavage product is
generated when Botulinum toxin serotype E is present in said
sample; (b) contacting said treated sample with a second partner of
the affinity couple, thereby forming stable complexes comprising
said first and second partners of said affinity couple; and (c)
assaying the presence or amount of said fluorescent cleavage
product in said treated sample, thereby determining Botulinum toxin
serotype E protease activity.
2. The method of claim 1, wherein said fluorescent protein is
selected from the group green fluorescent protein (GFP), blue
fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow
fluorescent protein (YFP) and red fluorescent protein (RFP).
3. The method of claim 2, wherein said fluorescent protein is
GFP.
4. The method of claim 1, 2 or 3, wherein said first partner of the
affinity couple is selected from the group histidine tag,
glutathione-S-transferase, maltose-binding protein, a biotinylation
sequence, streptavidin, S peptide, S protein, FLAG, hemagluttinin
(HA), c myc and AU1.
5. The method of claim 1, wherein said first partner of the
affinity couple is a histidine tag.
6. The method of claim 1, wherein said Botulinum toxin serotype E
recognition sequence comprises at least six consecutive residues of
SNAP-25, said six consecutive residues comprising Arg-Ile, or a
peptidomimetic thereof.
7. The method of claim 6, wherein said Botulinum toxin serotype E
recognition sequence comprises SEQ ID NO: 8, or a peptidomimetic
thereof.
8. The method of claim 6, wherein said Botulinum toxin serotype E
recognition sequence comprises residues 134 to 206 of SEQ ID NO: 2,
residues 137 to 206 of SEQ ID NO: 2, or a peptidomimetic
thereof.
9. The method of claim 1, wherein said substrate is cleaved with an
activity of at least 1 nanomole/minute/milligram toxin.
10. The method of claim 1, wherein said substrate is cleaved with
an activity of at least 100 nanomoles/minute/milligram toxin.
11. The method of claim 1, wherein said substrate is cleaved with
an activity of at least 1000 nanomoles/minute/milligram toxin.
12. The method of claim 1, wherein said second partner of the
affinity couple is immobilized.
13. The method of claim 1, wherein said second partner of the
affinity couple comprises cobalt (Co2+).
14. The method of claim 1, wherein said second partner of the
affinity couple comprises nickel (Ni2+).
15. The method of claim 1, further comprising separating said
fluorescent cleavage product from said stable complexes prior to
step (c).
16. The method of claim 15, wherein said separating comprises
applying said treated sample to a column, wherein said second
partner of the affinity couple is immobilized on said column.
17. The method of claim 15, wherein said separating comprises
applying said treated sample to a filter plate, wherein said second
partner of the affinity couple is immobilized on said filter
plate.
18. The method of claim 1, further comprising step (d) assaying the
amount of uncleaved tagged toxin substrate in said treated
sample.
19. The method of claim 1, wherein said sample is isolated
Botulinum toxin serotype E.
20. The method of claim 1, wherein said sample is isolated
Botulinum toxin serotype E light chain.
21. The method of claim 1, wherein said formulated product is a
formulated Botulinum toxin serotype E product.
22. The method of claim 1, wherein said sample is a whole or
partially purified cellular extract containing recombinantly
expressed Botulinum toxin serotype E.
Description
[0001] This application is a divisional and claims priority
pursuant to 35 U.S.C. .sctn. 120 to U.S. patent application Ser.
No. 10/917,844, filed Aug. 13, 2004, a continuation application
that claims priority pursuant to 35 U.S.C. .sctn. 120 to U.S.
patent application Ser. No. 09/942,098, filed Aug. 28, 2001, each
of which is hereby incorporated by reference in its entirety.
[0002] The present invention relates generally to protease assays,
and more specifically, to recombinantly produced substrates and
methods for assaying protease activity of clostridial toxins such
as botulinum toxins and tetanus toxins.
[0003] The neuroparalytic syndrome of tetanus and the rare but
potentially fatal disease, botulism, are caused by neurotoxins
produced by bacteria of the genus Clostridium. These clostridial
neurotoxins are highly potent and specific poisons of neural cells,
with the human lethal dose of the botulinum toxins on the order of
micrograms. Thus, the presence of even minute levels of botulinum
toxins in foodstuffs represents a public health hazard that must be
avoided through rigorous testing.
[0004] However, in spite of their potentially deleterious effects,
low controlled doses of botulinum neurotoxins have been
successfully used as therapeutics. These toxins have been used in
the therapeutic management of a variety of focal and segmental
dystonias, of strabismus and other conditions in which reversible
depression of a cholinergic nerve terminal activity is desired.
Established therapeutic uses of botulinum neurotoxins in humans
include, for example, treatment of blepharospasm, hemifacial spasm,
laringeal dysphonia, focal hyperhidrosis, hypersalivation,
oromandibular dystonia, cervical dystonia, torticollis, strabismus,
limbs dystonia, occupational cramps and myokymia (Rossetto et al.,
Toxicon 39:27-41 (2001)). Intramuscular injection of spastic tissue
with small quantities of BoNT/A, for example, has been used
effectively to treat spasticity due to brain injury, spinal cord
injury, stroke, multiple sclerosis and cerebral palsy. Additional
possible clinical uses of clostridial neurotoxins currently are
being investigated.
[0005] Given the potential danger associated with small quantities
of botulinum toxins in foodstuffs and the need to prepare accurate
pharmaceutical formulations, assays for botulinum neurotoxins
presently are employed in both the food and pharmaceutical
industry. The food industry requires assays for botulinum
neurotoxins in order to validate new food packaging methods and to
ensure food safety. In addition, the growing clinical use of the
botulinum toxins necessitates accurate assays for botulinum
neurotoxin activity for product formulation as well as quality
control. In both industries, a mouse lethality test currently is
used to assay for botulinum neurotoxin activity. Unfortunately,
this assay suffers from several drawbacks: cost due to the large
numbers of laboratory animals required; lack of specificity; and
the potential for inaccuracy unless large animal groups are
used.
[0006] Thus, there is a need for new materials and methods for
assaying for clostridial toxin protease activity. The present
invention satisfies this need and provides related advantages as
well.
[0007] The present invention provides methods of determining
clostridial toxin protease activity by (a) treating with a sample,
in solution phase under conditions suitable for clostridial toxin
protease activity, a tagged toxin substrate containing (i) a
fluorescent protein; (ii) a first partner of an affinity couple;
and (iii) a clostridial toxin recognition sequence that includes a
cleavage site which intervenes between the fluorescent protein and
the first partner of the affinity couple, such that a fluorescent
cleavage product is generated when clostridial toxin is present in
the sample; (b) contacting the treated sample with a second partner
of the affinity couple, thereby forming stable complexes containing
the first and second partners of the affinity couple; and (c)
assaying the presence or amount of the fluorescent cleavage product
in the treated sample, thereby determining clostridial toxin
protease activity. In one embodiment, the fluorescent cleavage
product is separated from the stable complexes prior to assaying
the presence or amount of the fluorescent cleavage product.
[0008] The present invention also provides a nucleic acid molecule
containing a nucleotide sequence that encodes a SNAP-25 substrate
which includes (i) a green fluorescent protein; (ii) a first
partner of an affinity couple; and (iii) a portion of SNAP-25 that
includes a BoNT/A, BoNT/C1 or BoNT/E recognition sequence
containing a cleavage site which intervenes between the green
fluorescent protein and the first partner of the affinity couple.
The present invention additionally provides a nucleic acid molecule
containing a nucleotide sequence that encodes a tagged toxin
substrate which includes (i) a fluorescent protein; (ii) a first
partner of an affinity couple; and (iii) a clostridial toxin
recognition sequence containing a cleavage site that intervenes
between the fluorescent protein and the first partner of the
affinity couple.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows a schematic of the deduced structure and
postulated mechanism of activation of clostridial neurotoxins.
Toxins can be produced as a single polypeptide chain of 150 kDa
which is composed of three 50 kDa domains connected by loops.
Selective proteolytic cleavage activates the toxins by generating
two disulfide-linked chains: the L chain of 50 kDa and the H chain
of 100 kDa, which is made up of two domains denoted H.sub.N and
H.sub.C. The three domains play distinct roles: the C-terminal
domain of the heavy chain (H.sub.C) functions in cell binding while
the N-terminal domain of the heavy chain (H.sub.N) permits
translocation from endosome to cell cytoplasm. Following reduction
of the disulfide linkage inside the cell, the zinc-endopeptidase
activity of the L chain is liberated.
[0010] FIG. 2 shows a schematic of the four steps required for
tetanus and botulinum toxin activity in central and peripheral
neurons.
[0011] FIG. 3 shows the subcellular localization at the plasma
membrane and sites of cleavage of SNAP-25, VAMP and syntaxin. VAMP
is bound to synaptic vesicle membrane, whereas SNAP-25 and syntaxin
are bound to the target plasma membrane. BoNT/A and /E cleave
SNAP-25 close to the carboxy-terminus, releasing nine or 26
residues, respectively. BoNT/B, /D, /F, /G and TeNT act on the
conserved central portion of VAMP (dotted) and release the
amino-terminal portion of VAMP into the cytosol. BoNT/C1 cleaves
SNAP-25 close to the carboxy-terminus as well as cleaving syntaxin
at a single site near the cytosolic membrane surface. The action of
BoNT/B, /C1, /D, /F, /G and TeNT results in release of a large
portion of the cytosolic domain of VAMP or syntaxin, while only a
small portion of SNAP-25 is released by selective proteolysis by
BoNT/A, /C1 or /E.
[0012] FIG. 4 shows the neurotoxin recognition motif of VAMP,
SNAP-25 and syntaxin. (A) Hatched boxes indicate the presence and
positions of a motif common to the three targets of clostridial
neurotoxins. (B) The recognition motif is composed of hydrophobic
residues ("h"); negatively charged Asp or Glu residues ("-") and
polar residues ("p"); "x" represents any amino acid. The motif is
included in regions of VAMP, SNAP-25 and syntaxin predicted to
adopt an .alpha.-helical conformation. (C) A top view of the motif
in an .alpha.-helical conformation is shown. Negatively charged
residues align on one face, while hydrophobic residues align on a
second face.
[0013] FIG. 5 shows an alignment of various SNAP-25 proteins. Human
SNAP-25 (SEQ ID NO: 2; GenBank accession g4507099; see, also,
related human SNAP-25 sequence g2135800); mouse SNAP-25 (SEQ ID NO:
12; GenBank accession G6755588); Drosophila SNAP-25 (SEQ ID NO: 13;
GenBank accession g548941); goldfish SNAP-25 (SEQ ID NO: 14;
GenBank accession g2133923); sea urchin SNAP-25 (SEQ ID NO: 15;
GenBank accession g2707818) and chicken SNAP-25 (SEQ ID NO: 16;
GenBank accession g481202) are depicted.
[0014] FIG. 6 shows an alignment of various VAMP proteins. Human
VAMP-1 (SEQ ID NO: 96; GenBank accession g135093); human VAMP-2
(SEQ ID NO: 4; GenBank accession g135094); mouse VAMP-2 (SEQ ID NO:
17; GenBank accession g2501081); bovine VAMP (SEQ ID NO: 18;
GenBank accession g89782); frog VAMP (SEQ ID NO: 19; GenBank
accession g6094391); and sea urchin VAMP (SEQ ID NO: 20; GenBank
accession g5031415) are depicted.
[0015] FIG. 7 shows an alignment of various syntaxin proteins.
Human syntaxin 1A (SEQ ID NO: 21; GenBank accession g15079184),
human syntaxin 1B2 (SEQ ID NO: 22; GenBank accession g15072437),
mouse syntaxin 1A (SEQ ID NO: 23; GenBank accession g15011853),
Drosophila syntaxin 1A (SEQ ID NO: 24; GenBank accession g2501095);
C. elegans syntaxin A (SEQ ID NO: 25; GenBank accession g7511662)
and sea urchin syntaxin (SEQ ID NO: 26; GenBank accession
g13310402) are depicted.
[0016] FIG. 8 shows (A) a schematic of plasmid pQBI
GFP-SNAP25.sub.(134-206) and (B) the nucleic acid and amino acid
sequences (SEQ ID NOS: 85 and 86) of
GFP-SNAP25.sub.(134-206)-6.times.HIS.
[0017] FIG. 9 shows (A) a schematic of plasmid pQBI
SNAP25.sub.(134-206)-GFP and (B) the nucleic acid and amino acid
sequences (SEQ ID NOS: 87 and 88) of
6.times.HIS-SNAP25.sub.(134-206)-GFP.
[0018] FIG. 10 shows SDS-PAGE and Western blot analysis of rLC/A
and BoNT/E proteolysis reactions. A, B, and C show rLC/A
proteolytic reactions, while D, E, and F show BoNT/E proteolytic
reactions. (A) Sypro Ruby stained SDS-PAGE of samples incubated
with rLC/A for 0, 5, 10, 15, 30, and 60 minutes. (B) Western blot
probing with anti-GFP primary antibody. (C) Western blot probing
with an antibody specific to the C-terminus of the
SNAP25.sub.197proteolysis product. The protein bands were
identified as 206 for the complete GFP-SNAP25.sub.(134-206) moiety
and as 197 for rLC/A-processed GFP-SNAP25.sub.(134-197). (D) Sypro
Ruby stained SDS-PAGE of samples incubated with BoNT/E for 0, 5,
10, 15, 30, and 60 minutes. (E) Western blot probing with anti-GFP
primary antibody. (F) Western blot probing with an antibody
specific to the C-terminus of the SNAP25.sub.180proteolysis
product. The protein bands identified as 206 represent the complete
GFP-SNAP25.sub.(134-206) moiety and those identified as 180
represent BoNT/E-processed GFP-SNAP25.sub.(134-180). (G) Schematic
summary of the GFP-SNAP fluorescence release assay.
[0019] FIG. 11 shows endopeptidase activity of recombinant light
chain, native and bulk A toxin. (A) Endopeptidase activity of
recombinant type A light chain. (B) Endopeptidase activity of
native BoNT/A dichain toxin. (C) Endopeptidase activity of bulk A
toxin.
[0020] FIG. 12 shows endopeptidase activity of native BoNT/E single
chain, native BoNT/E dichain and BoNT/C complex. (A) Endoprotease
activity of native single chain BoNT/E toxin. (B) Endoprotease
activity of native BoNT/E dichain (DC). (C) Endopeptidase activity
of BoNT/C complex.
[0021] FIG. 13 shows proteolysis of the GFP-SNAP25.sub.(131-206)
fusion protein substrate as well as substrate analogues containing
mutations R198A and R180D in the scissile bonds.
[0022] FIG. 14 shows proteolysis of fusion protein substrates using
crude cell lysates. (A) CODON PLUS.RTM. cell lysates. (B) Negative
control TOP10.RTM. cell lysates.
[0023] FIG. 15 shows representative examples of data collected for
kinetic analysis of rLC/A. The graph on the left shows the
non-linear curves fit to data collected over the course of 7-hour
reactions. The graph on the right shows the initial, linear
segments of the non-linear plots; the slopes of these lines are the
initial reaction rates at the specified substrate concentrations
(RFU/min).
[0024] FIG. 16 shows a plot of preliminary data for 178 pM rLC/A
activity, indicating that the K.sub.m is approximately 4.6
.mu.M.
[0025] FIG. 17 shows a GFP-SNAP25 assay of two vials of BOTOX.RTM.
(Botulinum toxin serotype A)
DETAILED DESCRIPTION
[0026] The invention provides nucleic acid molecules containing
nucleotide sequences encoding SNAP-25 substrates and tagged toxin
substrates useful for determining clostridial toxin protease
activity, including botulinum toxins of all serotypes as well as
tetanus toxins. The nucleic acid molecules of the invention are
valuable, in part, because they can be used to conveniently prepare
recombinant SNAP-25 substrates as well as tagged toxin substrates
with a longer toxin recognition sequence, which can enhance binding
affinity for the cognate clostridial toxin. Such recombinant
SNAP-25 substrates and tagged toxin substrates can be utilized in
simple screening assays which do not rely on animals and are useful
for analyzing crude and bulk samples as well as highly purified
dichain toxins or isolated clostridial toxin light chains.
Furthermore, as disclosed herein, recombinant SNAP-25 substrates
and tagged toxin substrates prepared from the nucleic acid
molecules of the invention can be used to detect BoNT/A and BoNT/E
at low picomolar concentrations, and to detect BoNT/C at low
nanomolar concentrations.
[0027] The present invention further provides methods of
determining clostridial toxin protease activity which are
advantageous in that they can be sensitive, rapid and
high-throughput and allow a solution phase proteolysis reaction.
Unlike other assays, the methods of the invention combine analysis
of a clostridial toxin substrate which has good affinity for its
cognate toxin, resulting in an assay with high sensitivity, in a
format in which the toxin protease activity is assayed in solution
phase, allowing kinetic analyses of toxin activity. Alternative
assays, such as those described in U.S. Pat. No. 6,762,280, have
relied on an immobilized substrate, albeit one with good binding
affinity for toxin. Additional assays have relied on high pressure
liquid chromatography (HPLC) separation and, therefore, have not
been amenable to a high-throughput format (U.S. Pat. No.
5,965,699), or have been lower sensitivity assays which relied on
short peptide substrates with relatively poor binding
characteristics (see, for example, Anne et al., Anal. Biochem. 291:
253-261 (2001)).
[0028] Thus, the present invention provides, in part, a nucleic
acid molecule containing a nucleotide sequence that encodes a
SNAP-25 substrate containing (i) a green fluorescent protein; (ii)
a first partner of an affinity couple; and (iii) a portion of
SNAP-25 which includes a BoNT/A, BoNT/C1 or BoNT/E recognition
sequence containing a cleavage site, where the cleavage site
intervenes between the green fluorescent protein and the first
partner of the affinity couple. In a nucleic acid molecule of the
invention, the encoded first partner of the affinity couple can be,
without limitation, a histidine tag, glutathione-S-transferase,
maltose-binding protein, biotinylation sequence, streptavidin, S
peptide, S protein, or an epitope such as a FLAG, hemagluttinin,
c-myc or AU1 epitope. In one embodiment, the encoded first partner
of the affinity couple is a histidine tag.
[0029] In a nucleic acid molecule of the invention, the encoded
SNAP-25 substrate can include any of a variety of portions of
SNAP-25 which have a BoNT/A, BoNT/C1 or BoNT/E recognition sequence
containing a cleavage site. Such a portion of SNAP-25 can include,
for example, residues 134 to 206 of SEQ ID NO: 90 or another
BoNT/A, BoNT/C1 or BoNT/E recognition sequence and cleavage site
disclosed herein or known in the art. In one embodiment, a nucleic
acid molecule of the invention includes a nucleotide sequence
encoding a SNAP-25 substrate which is cleaved with an activity of
at least 1 nanomole/minute/milligram toxin. In another embodiment,
a nucleic acid molecule of the invention includes a nucleotide
sequence encoding a SNAP-25 substrate which is cleaved with an
activity of at least 100 nanomoles/minute/milligram toxin. In a
further embodiment, a nucleic acid molecule of the invention
includes a nucleotide sequence encoding a SNAP-25 substrate which
is cleaved with an activity of at least 1000
nanomoles/minute/milligram toxin.
[0030] The present invention further provides a nucleic acid
molecule containing a nucleotide sequence encoding a tagged toxin
substrate that contains (i) a fluorescent protein; (ii) a first
partner of an affinity couple; and (iii) a clostridial toxin
recognition sequence containing a cleavage site, where the cleavage
site intervenes between the fluorescent protein and the first
partner of the affinity couple. In a nucleic acid molecule encoding
a tagged toxin substrate, the fluorescent protein can be, without
limitation, a green fluorescent protein, blue fluorescent protein,
cyan fluorescent protein, yellow fluorescent protein or red
fluorescent protein. In one embodiment, a nucleic acid molecule of
the invention includes a nucleotide sequence encoding a green
fluorescent protein.
[0031] In such a nucleic acid molecule, a variety of first partners
of an affinity couple can be incorporated into the encoded tagged
toxin substrate. As non-limiting examples, an encoded tagged toxin
substrate can include a histidine tag, glutathione-S-transferase,
maltose-binding protein, biotinylation sequence, streptavidin, S
peptide, S protein, or an epitope such as a FLAG, hemagluttinin,
c-myc or AU1 epitope as the first partner of the affinity couple.
Furthermore, the encoded clostridial toxin recognition sequence can
be, without limitation, a portion of SNAP-25 such as residues 134
to 206 of SEQ ID NO: 90; or a BoNT/A, BoNT/B, BoNT/C1, BoNT/D,
BoNT/E, BoNT/F, BoNT/G or TeNT recognition sequence such as, for
example, one of the recognition sequences disclosed herein or known
in the art.
[0032] Furthermore, a nucleic acid molecule of the invention
contains a nucleotide sequence encoding a tagged toxin substrate
which can be cleaved by cognate clostridial toxin with low or high
activity. In one embodiment, a nucleic acid molecule of the
invention encodes a tagged toxin substrate which can be cleaved
with an activity of at least 1 nanomole/minute/milligram toxin. In
another embodiment, a nucleic acid molecule of the invention
encodes a tagged toxin substrate which can be cleaved with an
activity of at least 100 nanomoles/minute/milligram toxin. In yet
another embodiment, a nucleic acid molecule of the invention
encodes a tagged toxin substrate which can be cleaved with an
activity of at least 1000 nanomoles/minute/milligram toxin.
[0033] The invention additionally provides a nucleic acid molecule
that contains a nucleotide sequence encoding a tagged toxin
substrate that includes (i) a genetically encoded detectable
marker; (ii) a first partner of an affinity couple; and (iii) a
clostridial toxin recognition sequence containing a cleavage site,
where the cleavage site intervenes between the genetically encoded
detectable marker and the first partner of the affinity couple. In
a nucleic acid molecule of the invention, the genetically encoded
detectable marker can be, without limitation, luciferase,
horseradish peroxidase, alkaline phosphatase or a fluorescent
protein.
[0034] Any of a variety of first partners of an affinity couple can
be combined with a genetically encoded detectable marker in a
tagged toxin substrate encoded by a nucleic acid molecule of the
invention. The encoded first partner of the affinity couple can be,
for example, a histidine tag; glutathione-S-transferase;
maltose-binding protein; biotinylation sequence such as BirAsp;
streptavidin; S peptide; S protein; or an epitope such as a FLAG,
hemagluttinin, c-myc or AU1 epitope. In one embodiment, a nucleic
acid molecule of the invention encodes a tagged toxin substrate
which includes a histidine tag as the first partner of the affinity
couple.
[0035] Furthermore, any of a variety of encoded clostridial toxin
recognition sequences can be combined with a genetically encoded
detectable marker in a tagged toxin substrate encoded by a nucleic
acid molecule of the invention. Such clostridial toxin recognition
sequences include, yet are not limited to, botulinum toxin
recognition sequences. As non-limiting examples, a clostridial
toxin recognition sequence to be combined with a genetically
encoded detectable marker in an encoded tagged toxin substrate can
be a portion of SNAP-25 such as residues 134 to 206 of SEQ ID NO:
90; or a BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or
TeNT recognition sequences such as one of the recognition sequences
disclosed herein or known in the art.
[0036] A nucleic acid molecule of the invention can encode a tagged
toxin substrate which is cleaved by cognate clostridial toxin with
low or high activity. In one embodiment, a nucleic acid molecule of
the invention encodes a tagged toxin substrate which can be cleaved
with an activity of at least 1 nanomole/minute/milligram toxin. In
other embodiments, a nucleic acid molecule of the invention encodes
a tagged toxin substrate which can be cleaved with an activity of
at least 100 nanomoles/minute/milligram toxin or at least 1000
nanomoles/minute/milligram toxin.
[0037] Further provided herein is a SNAP-25 substrate which
includes (i) a green fluorescent protein; (ii) a first partner of
an affinity couple; and (iii) a portion of SNAP-25 that includes a
BoNT/A, BoNT/C1 or BoNT/E recognition sequence containing a
cleavage site, where the cleavage site intervenes between the green
fluorescent protein and the first partner of the affinity couple.
Any of a variety of first partners of an affinity couple are useful
in a SNAP-25 substrate of the invention. As non-limiting examples,
a first partner of an affinity couple can be a histidine tag,
glutathione-S-transferase, maltose-binding protein, a biotinylation
sequence, streptavidin, S peptide, S protein, or an epitope such as
a FLAG, hemagluttinin, c-myc or AU1 epitope. In one embodiment, the
invention provides a SNAP-25 substrate in which the first partner
of the affinity couple is a histidine tag.
[0038] A SNAP-25 substrate of the invention incorporates a portion
of SNAP-25 which includes a BoNT/A, BoNT/C1 or BoNT/E recognition
sequence containing the corresponding cleavage site. In one
embodiment, a SNAP-25 substrate of the invention includes residues
134 to 206 of SEQ ID NO: 90. In further embodiments, a SNAP-25
substrate of the invention includes a BoNT/A recognition sequence,
a BoNT/C1 recognition sequence, or a BoNT/E recognition sequence.
Furthermore, a SNAP-25 substrate of the invention can be cleaved,
without limitation, with an activity of at least 1
nanomole/minute/milligram toxin, at least 100
nanomoles/minute/milligram toxin, or at least 1000
nanomoles/minute/milligram toxin.
[0039] Further provided herein is a tagged toxin substrate which
includes (i) a fluorescent protein; (ii) a first partner of an
affinity couple; and (iii) a clostridial toxin recognition sequence
containing a cleavage site, where the cleavage site intervenes
between the fluorescent protein and the first partner of the
affinity couple. Any of a variety of fluorescent proteins can be
incorporated into a tagged toxin substrate of the invention,
including, without limitation, green fluorescent proteins (GFPs),
blue fluorescent proteins (BFPs), cyan fluorescent proteins (CFPs),
yellow fluorescent proteins (YFPs) and red fluorescent proteins
(RFPs). In one embodiment, a tagged toxin substrate of the
invention includes a green fluorescent protein. Any of a variety of
first partners of an affinity couple are useful in the tagged toxin
substrates of the invention. As non-limiting examples, a tagged
toxin substrate can include a histidine tag,
glutathione-S-transferase, maltose-binding protein, a biotinylation
sequence, streptavidin, S peptide, S protein, or an epitope such as
a FLAG, hemagluttinin, c-myc or AU1 epitope as the first partner of
the affinity couple. In one embodiment, the invention provides a
tagged toxin substrate in which the first partner of the affinity
couple is a histidine tag.
[0040] It is understood that a variety of recognition sequences are
useful in the tagged toxin substrates of the invention, including,
yet not limited to, botulinum toxin recognition sequences. In one
embodiment, the invention provides a tagged toxin substrate in
which the recognition sequence includes a portion of SNAP-25 such
as, without limitation, residues 134 to 206 of SEQ ID NO: 90. In
another embodiment, the invention provides a tagged toxin substrate
in which the recognition sequence is a BoNT/A recognition sequence
such as, without limitation, a BoNT/A recognition sequence
including at least six consecutive residues of SNAP-25, where the
six consecutive residues encompass the sequence Gln-Arg. In a
further embodiment, the invention provides a tagged toxin substrate
in which the recognition sequence is a BoNT/B recognition sequence
such as, without limitation, a BoNT/B recognition sequence which
includes at least six consecutive residues of VAMP, where the six
consecutive residues encompass the sequence Gln-Phe. In still
another embodiment, the invention provides a tagged toxin substrate
in which the recognition sequence is a BoNT/C1 recognition sequence
such as, without limitation, a BoNT/C1 recognition sequence which
includes at least six consecutive residues of syntaxin, where the
six consecutive residues encompass the sequence Lys-Ala, or a
BoNT/C1 recognition sequence which includes at least six
consecutive residues of SNAP-25, where the six consecutive residues
encompass the sequence Arg-Ala. In still another embodiment, the
invention provides a tagged toxin substrate in which the
recognition sequence is a BoNT/D recognition sequence such as,
without limitation, a BoNT/D recognition sequence including at
least six consecutive residues of VAMP, where the six consecutive
residues encompass the sequence Lys-Leu.
[0041] In yet another embodiment, the invention provides a tagged
toxin substrate in which the recognition sequence is a BoNT/E
recognition sequence such as, without limitation, a BoNT/E
recognition sequence which includes at least six consecutive
residues of SNAP-25, the six consecutive residues encompassing the
sequence Arg-Ile. In a further embodiment, the invention provides a
tagged toxin substrate in which the recognition sequence is a
BoNT/F recognition sequence such as, without limitation, a BoNT/F
recognition sequence including at least six consecutive residues of
VAMP, the six consecutive residues encompassing the sequence
Gln-Lys. The present invention additionally provides a tagged toxin
substrate in which the recognition sequence is a BoNT/G recognition
sequence such as, without limitation, a BoNT/G recognition sequence
including at least six consecutive residues of VAMP, where the six
consecutive residues encompass the sequence Ala-Ala. In still
another embodiment, the invention provides a tagged toxin substrate
in which the recognition sequence is a TeNT recognition sequence
such as, without limitation, a TeNT recognition sequence which
includes at least six consecutive residues of VAMP, where the six
consecutive residues encompass the sequence Gln-Phe.
[0042] A tagged toxin substrate of the invention can be cleaved
with high or low activity. In one embodiment, a tagged toxin
substrate of the invention can be cleaved with an activity of at
least 1 nanomole/minute/milligram toxin. In another embodiment, a
tagged toxin substrate of the invention can be cleaved with an
activity of at least 100 nanomoles/minute/milligram toxin. In still
a further embodiment, a tagged toxin substrate of the invention can
be cleaved with an activity of at least 1000
nanomoles/minute/milligram toxin.
[0043] Tetanus and botulinum neurotoxins are produced by Clostridia
and cause the neuroparalytic syndromes of tetanus and botulism.
While tetanus neurotoxin acts mainly at the CNS synapse, botulinum
neurotoxins act peripherally. Clostridial neurotoxins share a
similar mechanism of cell intoxication, blocking the release of
neurotransmitters. In these toxins, which are composed of two
disulfide-linked polypeptide chains, the larger subunit is
responsible for neurospecific binding and translocation of the
smaller subunit into the cytoplasm. Upon translocation and
reduction in neurons, the smaller chain displays protease activity
specific for protein components involved in neuroexocytosis in the
neuronal cytosol. The SNARE protein targets of clostridial toxins
are common to exocytosis in a variety of non-neuronal types; in
these cells, as in neurons, light chain protease activity inhibits
exocytosis.
[0044] Tetanus neurotoxin and botulinum neurotoxins B, D, F, and G
specifically recognize VAMP (synaptobrevin), an integral protein of
the synaptic vesicle membrane which is cleaved at distinct bonds
depending on the neurotoxin. Botulinum A and E neurotoxins
recognize and specifically cleave SNAP-25, a protein of the
presynaptic membrane, at two different sites in the
carboxy-terminal portion of the protein. Botulinum neurotoxin C1
cleaves syntaxin, a protein of the nerve plasmalemma, in addition
to SNAP-25. The three protein targets of the Clostridial
neurotoxins are conserved from yeast to humans, although cleavage
sites and toxin susceptibility are not necessarily conserved (see
below; see, also, Humeau et al., Biochimie 82:427-446 (2000);
Niemann et al., Trends in Cell Biol. 4:179-185 (1994); and
Pellizzari et al., Phil. Trans. R. Soc. London 354:259-268
(1999)).
[0045] Naturally occurring tetanus and botulinum neurotoxins are
produced as polypeptide chains of 150 kDa without a leader
sequence. These toxins may be cleaved by bacterial or tissue
proteinases at an exposed protease-sensitive loop, generating
active di-chain toxin. Naturally occurring clostridial toxins
contain a single interchain disulfide bond bridging the heavy chain
(H, 100 kDa) and light chain (L, 50 kDa); such a bridge is
important for neurotoxicity of toxin added extracellularly
(Montecucco and Schiavo, Quarterly Rev. Biophysics 28:423-472
(1995)).
[0046] The clostridial toxins appear to be folded into three
distinct 50 kDa domains, as shown in FIG. 1, with each domain
having a separate functional role. As illustrated in FIG. 2, the
cell intoxication mechanism of the clostridial toxins consists of
four distinct steps: (1) binding; (2) internalization; (3) membrane
translocation; and (4) enzymatic target modification. The
carboxy-terminal part of the heavy chain (H.sub.C) functions in
neurospecific binding, while the amino-terminal portion of the H
chain (H.sub.N) functions in membrane translocation. The L chain is
responsible for the intracellular catalytic activity (Montecucco
and Schiavo, supra, 1995).
[0047] The amino acid sequences of eight human clostridial
neurotoxins have been derived from the corresponding genes
(Neimann, "Molecular Biology of Clostridial Neurotoxins" in
Sourcebook of Bacterial Protein Toxins Alouf and Freer (Eds.) pp.
303-348 London: Academic Press 1991). The L and H chains are
composed of roughly 439 and 843 residues, respectively, and
homologous segments are separated by regions of little or no
similarity. The most well conserved regions of the L chains are the
amino-terminal portion (100 residues) and central region
(corresponding to residues 216 to 244 of TeNT), as well as the two
cysteines forming the interchain disulfide bond. The 216 to 244
region contains a His-Glu-X-X-His binding motif characteristic of
zinc-endopeptidases.
[0048] The heavy chain is less well conserved than the light chain,
with the carboxy-terminal part of H.sub.C (corresponding to
residues 1140 to 1315 of TeNT) being the most variable. This is
consistent with the involvement of the H.sub.C domain in binding to
nerve terminals and the fact that the different neurotoxins appear
to bind different receptors. Not surprisingly, many serotype
specific antibodies recognize heavy chain determinants.
[0049] Comparison of the nucleotide and amino acid sequences of the
clostridial toxins indicates that they derive from a common
ancestral gene. Spreading of the clostridial neutrotoxin genes may
have been facilitated by the fact that these genes are located on
mobile genetic elements. As discussed further below, sequence
variants of the clostridial neurotoxins, including the seven
botulinum toxins are known in the art. See, for example, FIGS. 5 to
7 and Humeau et al., supra, 2000.
[0050] As discussed above, natural targets of the clostridial
neurotoxins include VAMP, SNAP-25, and syntaxin. As depicted in
FIG. 3, VAMP is associated with the synaptic vesicle membrane,
whereas SNAP-25 and syntaxin are associated with the target
membrane. BoNT/A and BoNT/E cleave SNAP-25 in the carboxy-terminal
region, releasing nine or twenty-six amino acid residues,
respectively, and BoNT/C1 also cleaves SNAP-25 near the
carboxy-terminus. The botulinum serotypes BoNT/B, BoNT/D, BoNT/F
and BoNT/G, and tetanus toxin, act on the conserved central portion
of VAMP, and release the amino-terminal portion of VAMP into the
cytosol. BoNT/C1 cleaves syntaxin at a single site near the
cytosolic membrane surface. Thus, the action of BoNT/B, BoNT/C1,
BoNT/D, BoNT/F, BoNT/G and TeNT results in release of a large
portion of the cytosolic domain of VAMP or syntaxin, while only a
small portion of SNAP-25 is released by proteolysis of BoNT/A,
BoNT/C1 or BoNT/E (Montecucco and Schiavo, supra, 1995).
[0051] SNAP-25, a protein of about 206 residues lacking a
transmembrane segment, is associated with the cytosolic surface of
the nerve plasmalemma (FIG. 3; see, also, Hodel et al., Int. J.
Biochemistry and Cell Biology 30:1069-1073 (1998)). In addition to
homologs highly conserved from Drosophila to mammals,
SNAP-25-related proteins also have been cloned from yeast. SNAP-25
is required for axonal growth during development and may be
required for nerve terminal plasticity in the mature nervous
system. In humans, two isoforms are differentially expressed during
development; isoform a is constitutively expressed beginning in the
embryo stage, while isoform b appears at birth and predominates in
adult life. SNAP-25 analogues such as SNAP-23 also are expressed
outside the nervous system, for example, in pancreatic cells.
[0052] VAMP is a protein of about 120 residues, with the exact
length depending on the species and isotype. As shown in FIG. 3,
VAMP contains a short carboxy-terminal segment inside the vesicle
lumen while most of the molecule is exposed to the cytosol. The
proline-rich amino-terminal thirty residues are divergent among
species and isoforms while the central portion of VAMP (residues 30
to 96), which is rich in charged and hydrophilic residues and
includes known cleavage sites, is highly conserved. VAMP is
associated on the synaptic vesicle membrane with synaptophysin.
[0053] A variety of species homologs of VAMP are known in the art
including, without limitation, human, rat, bovine, Torpedo,
Drosophila, yeast, squid and Aplysia homologs. In addition,
multiple isoforms of VAMP have been identified, including VAMP-1,
VAMP-2 and cellubrevin, and insensitive forms have been identified
in non-neuronal cells. VAMP appears to be present in all vertebrate
tissues although the distribution of VAMP-1 and VAMP-2 varies in
different cell types. Chicken and rat VAMP-1 are not cleaved by
TeNT or BoNT/B. These VAMP-1 homologs have a valine in place of
glutamine present in human and mouse VAMP-1 at the TeNT or BoNT/B
cleavage site. The substitution does not affect the activity of
BoNT/D, /F or /G, which cleave both VAMP-1 and VAMP-2 with similar
rates.
[0054] Syntaxin, located on the cytosolic surface of the nerve
plasmalemma, is membrane-anchored via a carboxy-terminal segment
such that most of the protein is exposed to the cytosol. Syntaxin
colocalizes with calcium channels at the active zones of the
presynaptic membrane where neurotransmitter release takes place. In
addition, syntaxin interacts with synaptotagmin, a protein of the
SSV membrane which forms a functional bridge between the
plasmalemma and vesicles. A variety of syntaxin isoforms have been
identified. Two isoforms of slightly different lengths (285 and 288
residues) have been identified in nerve cells (isoforms 1A and 1B),
with isoforms 2, 3, 4 and 5 present in other tissues. The isoforms
have varying sensitivities to BoNT/C1, with the 1A, 1B, 2 and 3
syntaxin isoforms cleaved by this toxin.
[0055] As indicated above, a SNAP-25 substrate of the invention
includes (i) a green fluorescent protein; (ii) a first partner of
an affinity couple; and (iii) a portion of SNAP-25 that includes a
BoNT/A, BoNT/C1 or BoNT/E recognition sequence containing a
cleavage site, where the cleavage site intervenes between the green
fluorescent protein and the first partner of the affinity couple. A
tagged toxin substrate of the invention includes (i) a fluorescent
protein; (ii) a first partner of an affinity couple; and (iii) a
clostridial toxin recognition sequence containing a cleavage site,
where the cleavage site intervenes between the fluorescent protein
and the first partner of the affinity couple.
[0056] A SNAP-25 substrate includes, in part, a green fluorescent
protein. As used herein, the term "green fluorescent protein" is
synonymous with "GFP" and means a protein which absorbs light of a
certain wavelength and emits light energy of wavelengths in the
range of 520-565 nm. Green fluorescent proteins useful in the
invention include, without limitation, wild type green fluorescent
proteins such as A. Victoria GFP (SEQ ID NO: 98) or homologs
thereof, as well as naturally occurring and genetically engineered
variants of wild type green fluorescent proteins, and active
fragments thereof that retain the ability to emit light in the
range of 520-565 nm. As non-limiting examples, the term "green
fluorescent protein" includes the Ser65Thr variant of GFP of wild
type A. Victoria GFP, which demonstrates accelerated fluorophore
formation (Heim et al., Nature 373:663 (1995)); GFP variants
containing a Ser65 to Thr, Ala, Gly, Cys or Leu substitution, which
convert the major and minor absorbance peaks of wild type GFP to a
single absorbance peak at 489 nm, producing brighter green
fluorescent proteins (Heim et al., supra, 1995); GFP variants such
as Phe64Leu, which alleviate the temperature sensitivity of wild
type GFP (Tsien et al., Biochem. 67:509 (1998)); Ala206Lys, Leu221
Lys, and Phe223Arg variants of GFP, which overcome dimerization at
high concentrations (Zacharias et al., Science 296:913 (2002)); and
enhanced GFP (EGFP), which combines codon optimization for
expression in mammalian cells with the Ser65Thr and Phe64Leu
substitutions, resulting in a bright, stable variant (Cormack et
al., Gene 173:33 (1996)). In one embodiment, a green fluorescent
protein useful in the invention has at least 70% amino acid
identity with the wild type A. Victoria GFP (SEQ ID NO: 98). In
other embodiments, a green fluorescent protein useful in the
invention has at least 75%, 80%, 85%, 90% or 95% amino acid
identity with the wild type A. Victoria GFP (SEQ ID NO: 98). In a
further embodiment, a green fluorescent protein useful in the
invention has at most ten amino acid substitutions relative to the
wild type A. Victoria GFP (SEQ ID NO: 98). In still further
embodiments, a green fluorescent protein useful in the invention
has at most one, two, three, four, five, six, seven, eight or nine
amino acid substitutions relative to the wild type A. Victoria GFP
(SEQ ID NO: 98).
[0057] A tagged toxin substrate includes, in part, a fluorescent
protein. As used herein, the term "fluorescent protein" means a
protein which absorbs light energy of a certain wavelength and
emits light energy of a longer wavelength. Fluorescent proteins
useful in the invention encompass, without limitation, wild type
fluorescent proteins and naturally occurring or genetically
engineered variants of fluorescent proteins such as those derived
from marine organisms.
[0058] Fluorescent proteins useful in tagged toxin substrates
include, without limitation, A. victoria-derived fluorescent
proteins (AFPs) such as wild type A. victoria proteins and
naturally occurring or genetically engineered variants of A.
victoria proteins. Such fluorescent proteins include, but are not
limited to, green fluorescent proteins (GFPs), cyan fluorescent
proteins (CFPs), blue fluorescent proteins (BFPs) and yellow
fluorescent proteins (YFPs), where the color of the fluorescence
depends on the wavelength of the emitted light; green fluorescent
proteins emit light in the range of 520-565 nm; cyan fluorescent
proteins emit light in the range of 500-520 nm; blue fluorescent
proteins emit light in the range of 450-500 nm; yellow fluorescent
proteins emit light in the range of 565-590 nm; and red fluorescent
proteins, described further below, emit light in the range of
625-740 nm. Furthermore, fluorescent proteins useful in the
invention include, for example, those which have been genetically
engineered for improved properties such as, without limitation,
altered excitation or emission wavelengths; enhanced brightness, pH
resistance, stability or speed of fluorophore formation;
photoactivation; or reduced oligomerization or photobleaching. A
fluorescent protein useful in the invention also can be engineered
for improved protein expression by converting wild type codons to
other codons more efficiently utilized in the cells which serve to
express the SNAP-25 or tagged toxin substrate which includes the
fluorescent protein.
[0059] Fluorescent proteins useful in the invention encompass those
which emit in a variety of spectra, including violet, blue, cyan,
green, yellow, orange and red. As described further below,
fluorescent proteins useful in the invention also include, yet are
not limited to, blue fluorescent proteins (BFPs) and cyan
fluorescent proteins (CFPs) produced by random mutagenesis of GFP
and rationally designed yellow fluorescent proteins (YFPs). BFP has
a Tyr66His substitution relative to GFP that shifts the absorbance
spectrum to a peak of 384 nm with emission at 448 nm (Heim et al.,
Proc. Natl. Acad. Sci. U.S.A. 91:12501 (1994)). CFP, which is
brighter and more photostable than BFP, has an absorption/emission
spectral range intermediate between BFP and EGFP due to a Tyr66Trp
substitution (Heim et al., supra, 1994; Heim and Tsien, Curr. Biol.
6:178-182 (1996); and Ellenberg et al., Biotechniques 25:838
(1998)); the Thr203Tyr CFP variant known as "CGFP" has excitation
and emission wavelengths intermediate between CFP and EGFP. The
rationally designed YFP has red-shifted absorbance and emission
spectra with respect to green fluorescent proteins (Ormo et al.,
Science 273:1392 (1996); Heim and Tsien, supra, 1996). A variety of
YFP variants display improved characteristics including, without
limitation, the YFP variants "Citrine" (YFP-Val68Leu/Gln69Met;
Griesbeck et al., J. Biol. Chem. 276:29188-29194 (2001)) and
"Venus" (YFP-Phe46Leu/Phe64Leu/Met153Thr/Val163Ala/Ser175Gly), an
extremely bright and fast-maturing YFP (Nagai et al., Nature
Biotech. 20:87-90 (2002)). One skilled in the art understands that
these and a variety of other fluorescent proteins which are
derived, for example, from GFP or other naturally occurring
fluorescent proteins also can be useful in the invention. See, for
example, Lippincott-Schwartz, Science 300:87 (2003), and Zhang et
al., Nature Reviews 3:906-918 (2002).
[0060] A fluorescent protein useful in the invention also can be a
long wavelength fluorescent protein such as a red or far-red
fluorescent protein, which can be useful for reducing or
eliminating background fluorescence from samples derived from
eukaryotic cells or tissues. Such red fluorescent proteins include
naturally occurring and genetically modified forms of Discosoma
striata proteins including, without limitation, DsRed (DsRed1 or
drFP583; Matz et al., Nat. Biotech. 17:969-973 (1999)); dsRed2
(Terskikh et al., J. Biol. Chem. 277:7633-7636 (2002)); T1
(dsRed-Express; Clontech; Palo Alto, Calif.; Bevis and Glick,
Nature Biotech. 20:83-87 (2002)); and the dsRed variant mRFP1
(Campbell et al., Proc. Natl. Acad. Sci. USA 99:7877-7882 (2002)).
Such red fluorescent proteins further include naturally occurring
and genetically modified forms of Heteractis crispa proteins such
as HcRed (Gurskaya et al., FEBS Lett. 507:16 (2001)).
[0061] Fluorescent proteins useful in a tagged toxin substrate can
be derived from any of a variety of species including marine
species such as A. victoria and other coelenterate marine
organisms. Useful fluorescent proteins encompass, without
limitation, Renilla mulleri-derived fluorescent proteins such as
the dimeric Renilla mulleri GFP, which has narrow excitation (498
nm) and emission (509 nm) peaks (Peele et al., J. Prot. Chem.
507-519 (2001)); Anemonia sulcata fluorescent proteins such as
DsRed proteins, for example, asFP595 (Lukyanov et al., J. Biol.
Chem. 275: 25879-25882 (2000)); Discosoma fluorescent proteins, for
example, Discosoma striata red fluorescent proteins such as dsFP593
(Fradkov et al., FEBS Lett. 479:127-130 (2000)); Heteractis crispa
fluorescent proteins such as HcRed and HcRed-2A (Gurskaya et al.,
FEBS Lett. 507:16-20 (2001)); and Entacmeae quadricolor fluorescent
proteins including red fluorescent proteins such as eqFP611
(Wiedenmann et al., Proc. Natl. Acad. Sci. USA 99:11646-11651
(2002)). One skilled in the art understands that these and many
other fluorescent proteins, including species homologs of the above
described naturally occurring fluorescent proteins as well as
engineered fluorescent proteins can be useful in recombinant tagged
toxin substrates encoded by nucleic acid molecules of the
invention. Expression vectors suitable for bacterial, mammalian and
other expression of fluorescent proteins are available from a
variety of commercial sources including BD Biosciences (Palo Alto,
Calif.).
[0062] As used herein, the term "fluorescent cleavage product"
means that portion of a tagged toxin substrate containing the
fluorescent protein, where the portion is generated by proteolysis
at the clostridial toxin cleavage site. By definition, a
"fluorescent cleavage product" does not include the first partner
of the affinity couple.
[0063] Further provided herein is a tagged toxin substrate which
includes a genetically encoded detectable marker. As used herein,
the term "genetically encoded detectable marker" means a protein
having a property such that the relative quantity of a substrate or
cleavage product containing the marker can be readily determined.
Such a genetically encoded detectable marker generates a detectable
cleavage product when the marker is included in a tagged toxin
substrate which is treated with a sample containing clostridial
toxin. Any of a variety of genetically encoded detectable markers
are useful in the invention including, but not limited to, enzymes;
tetracysteine motifs; fluorescent, bioluminescent, chemiluminescent
and other luminescent proteins; haptens; and single-chain
antibodies.
[0064] As used herein, the term "detectable cleavage product" means
that portion of a tagged toxin substrate containing a genetically
encoded detectable marker, where the portion is generated by
proteolysis of the tagged toxin substrate at the clostridial toxin
cleavage site.
[0065] One skilled in the art understands that the relative
quantity of a detectable cleavage product is determined using a
system or instrument appropriate to the genetically encoded
detectable marker. As examples, a spectrophotometer can be used to
assay a chromogenic detectable cleavage product generated from a
tagged toxin substrate containing a genetically encoded chromogenic
marker; a fluorometer can be used to assay a fluorescent detectable
cleavage product generated from a tagged toxin substrate containing
a genetically encoded fluorescent marker; and a luminometer can be
used to assay a luminescent detectable cleavage product generated
from a tagged toxin substrate containing a genetically encoded
luminescent marker.
[0066] Any of a variety of genetically encoded detectable markers
are useful in a tagged toxin substrate. In one embodiment, the
genetically encoded detectable marker is an enzyme such as, without
limitation, horseradish peroxidase (HRP), alkaline phosphatase
(AP), luciferase; .beta.-galactosidase, urease,
.beta.-glucouronidase (GUS), glucose oxidase or .beta.-lactamase.
As non-limiting examples, the relative quantity of a
horseradish-peroxidase containing detectable cleavage product can
be determined using a chromogenic substrate such as
tetramethylbenzidine (TMB), yielding a soluble product in the
presence of hydrogen peroxide which is detectable by measuring
absorbance at 450 nm; the relative quantity of a detectable
alkaline phosphatase-containing cleavage product can be determined
using a chromogenic substrate such as p-nitrophenyl phosphate,
which yields a soluble product readily detectable by measuring
absorbance at 405 nm; the relative quantity of a detectable
luciferase-containing cleavage product can be determined using
luciferin as a substrate in the presence of ATP, Mg.sup.2+ and
molecular oxygen (Bronstein et al., Anal. Biochem. 219:169-181
(1994)); and the relative quantity of a detectable
.beta.-galactosidase containing cleavage product can be determined
using a chromogenic substrate such as
o-nitrophenyl-.beta.-D-galactopyranoside (ONPG), which yields a
soluble product detectable by measuring absorbance at 410 nm, or
detectable by chemiluminescence using, for example, a 1,2-dioxetane
substrate (Bronstein et al., supra, 1994). Similarly, the relative
quantity of a detectable urease-containing cleavage product can be
determined using a substrate such as urea-bromocresol purple (Sigma
Immunochemicals, St. Louis, Mo.); and the relative quantity of a
detectable .beta.-glucouronidase (GUS)-containing cleavage product
can be determined with a colorimetric assay using, for example, a
.beta.-glucouronide substrate such as X-Gluc; with a fluorescence
assay using, for example, 4-methylumbelliferyl-.beta.-D-galactoside
(4-MUG; Jefferson et al., supra, 1987); or with a chemiluminescent
assay using, for example, an adamantyl 1,2-dioxetane aryl
glucuronide substrate (Bronstein et al., supra, 1994). In the same
fashion, the relative quantity of a detectable
.beta.-lactamase-containing cleavage product can be determined, for
example, with a fluorescence assay using, for example, a
fluorescent substrate ester (Zlokarnik et al., Science 279:84-88
(1998)). See, also, Ausubel, Current Protocols in Molecular Biology
John Wiley & Sons, Inc., New York 2000.
[0067] A genetically encoded detectable marker useful in a tagged
toxin substrate also can be a fluorescent protein such as, without
limitation, a naturally occurring or genetically engineered variant
of a fluorescent Aequorea victoria, Renilla mulleri, Anemonia
sulcata, Discosoma striata, Heteractis crispa, and Entacmeae
quadricolor fluorescent protein. A fluorescent protein useful in
the invention further can be, without limitation, a green
fluorescent protein, blue fluorescent protein, cyan fluorescent
protein, yellow fluorescent protein or red fluorescent protein. A
variety of fluorescent proteins useful in the invention are
described hereinabove and are otherwise known in the art. See, for
example, Zhang et al., supra, 2002; Falk, Trends Cell Biol.
12:399-404 (2002); Selvin, supra, 2000; and Mahajan et al., supra,
1999.
[0068] A genetically encoded detectable marker useful in the
invention also can be a tetracysteine motif. Exemplary
tetracysteine motifs useful in the invention include, without
limitation, the sequence Cys-Cys-Xaa-Xaa-Cys-Cys (SEQ ID NO: 99) or
Cys-Cys-Pro-Gly-Cys-Cys (SEQ ID NO: 100). When combined with a
biarsenical reagent, a reduced tetracysteine motif forms a
fluorescent complex in which each arsenic atom of the conjugate
cooperatively binds a pair of cysteines within the motif. Thus, the
relative quantity of a tetracysteine motif-containing cleavage
fragment can be determined by its ability to form a fluorescent
covalent complex when combined with a biarsenical protein such as
the resorufin-based red label (ReAsH-EDT.sub.2), the fluorescein
arsenical helix binder (FlAsH-EDT.sub.2) or the biarsenical protein
CHoXAsH-EDT.sub.2 (Adams et al., J. Am. Chem. Soc. 124:6063-6076
(2002), and Zhang et al., supra, 2002). It is understood that these
and other biarsenical proteins are useful for determining the
relative quantity of a tetracysteine-motif containing cleavage
fragment in a method of the invention.
[0069] A genetically encoded detectable marker useful in the
invention also can be a hapten or single-chain antibody. A variety
of genetically encoded haptens are known in the art, including, yet
not limited to, FLAG, hemagluttinin (HA), c-myc, 6-HIS and AU1
haptens, which can be detected in conjunction with commercially
available antibodies as disclosed hereinbelow. Using procedures
well known in the art, the relative quantity of a hapten-containing
detectable cleavage fragment can be determined using a labeled
anti-hapten antibody or labeled secondary antibody. As a
non-limiting example, an enzyme-linked immunosorbent assay (ELISA)
can be useful for determining the relative quantity of a
hapten-containing detectable cleavage product. One skilled in the
art understands that, where a tagged toxin substrate includes a
genetically encoded detectable marker which is a hapten, such a
hapten is selected to be distinct from the first and second
partners of the affinity couple. One skilled in the art further
understands that these and a variety of other well-known
genetically encoded detectable markers including, but not limited
to, enzymes; tetracysteine motifs; fluorescent, bioluminescent,
chemiluminescent and other luminescent proteins; haptens; and
single-chain antibodies can be useful in the tagged toxin
substrates of the invention.
[0070] A SNAP-25 or tagged toxin substrate includes a first partner
of an affinity couple. As used herein, the term "affinity couple"
means first and second partners which are capable of forming a
stable, non-covalent association. An affinity couple useful in the
invention can be, without limitation, a histidine tag-metal;
binding protein-ligand; biotinylation sequence-streptavidin;
streptavidin-biotin; S peptide-S protein; antigen-antibody; or
receptor-ligand.
[0071] As indicated above, a first partner of an affinity couple is
included in a SNAP-25 or tagged toxin substrate. In particular, a
SNAP-25 substrate contains a BoNT/A, /C1 or /E cleavage site which
intervenes between the green fluorescent protein and the first
partner of the affinity couple. Thus, upon proteolysis at the
cleavage site, the green fluorescent protein is separated from the
portion of the SNAP-25 substrate containing the first partner of
the affinity couple. Similarly, a tagged toxin substrate contains a
clostridial toxin cleavage site which intervenes between the
fluorescent protein or other genetically encoded detectable marker
and the first partner of the affinity couple. Thus, upon
proteolysis at the cleavage site of a tagged toxin substrate, the
fluorescent protein or genetically encoded detectable marker is
separated from the portion of the tagged toxin substrate that
contains the first partner of the affinity couple. As described
further below, the methods of the invention can be practiced by
contacting a treated sample with the second partner of the affinity
couple in order to separate the fluorescent or otherwise detectable
cleavage product (which lacks the first partner of the affinity
couple) from uncleaved substrate and other components of the
treated sample which contain the first partner of the affinity
couple.
[0072] A SNAP-25 or tagged toxin substrate includes a clostridial
toxin recognition sequence. As used herein, the term "clostridial
toxin recognition sequence" means a scissile bond together with
adjacent or non-adjacent recognition elements, or both, sufficient
for detectable proteolysis at the scissile bond by a clostridial
toxin under conditions suitable for clostridial toxin protease
activity.
[0073] In a SNAP-25 or tagged toxin substrate, a cleavage site
"intervenes" between a green fluorescent protein or other
fluorescent protein or genetically encoded detectable marker and
the first partner of the affinity couple. Thus, the cleavage site
is positioned in between the green fluorescent protein, or other
fluorescent protein or genetically encoded detectable marker, and
the first partner of the affinity couple, such that proteolysis at
the cleavage site results in a fluorescent or otherwise detectable
cleavage product, which lacks the first partner of the affinity
couple, and the remaining portion of the substrate, which includes
the first partner of the affinity couple. It is understood that all
or only a part of the clostridial toxin recognition sequence can
intervene between the green fluorescent protein or other
fluorescent protein or genetically encoded detectable marker and
the first partner of the affinity couple.
[0074] A SNAP-25 or tagged toxin substrate contains a clostridial
toxin cleavage site which is positioned between a green fluorescent
protein, or other fluorescent protein or genetically encoded
detectable marker, and a first partner of an affinity couple. In
one embodiment, the green fluorescent protein, or other fluorescent
protein or genetically encoded detectable marker, is positioned
amino-terminal of the cleavage site while the first partner of the
affinity couple is positioned carboxy-terminal of the cleavage
site. In another embodiment, the green fluorescent protein, or
other fluorescent protein or genetically encoded detectable marker,
is positioned carboxy-terminal of the cleavage site while the first
partner of the affinity couple is positioned amino-terminal of the
cleavage site.
[0075] Clostridial toxins have specific and distinct cleavage
sites. BoNT/A cleaves a Gln-Arg bond; BoNT/B and TeNT cleave a
Gln-Phe bond; BoNT/C1 cleaves a Lys-Ala or Arg-Ala bond; BoNT/D
cleaves a Lys-Leu bond; BoNT/E cleaves an Arg-Ile bond; BoNT/F
cleaves a Gln-Lys bond; and BoNT/G cleaves an Ala-Ala bond (see
Table 1). In standard nomenclature, the sequence surrounding a
clostridial toxin cleavage site is denoted
P.sub.5-P.sub.4-P.sub.3-P.sub.2-P.sub.1-P.sub.1'-P.sub.2'-P.sub.3'-P.sub.-
4'-P.sub.5', with P.sub.1-P.sub.1' representing the scissile bond.
It is understood that a P.sub.1 or P.sub.1' site, or both, can be
substituted with another amino acid or amino acid mimetic in place
of the naturally occurring residue. For example, BoNT/A substrates
have been prepared in which the P.sub.1 position (Gln) is modified
to be an alanine, 2-aminobutyric acid or asparagine residue, and
these substrates were hydrolyzed by BoNT/A at the P.sub.1-Arg bond
(Schmidt and Bostian, J. Protein Chem. 16:19-26 (1997)). While
substitutions can be introduced at the P.sub.1 position of the
scissile bond, for example, a BoNT/A scissile bond, it is further
recognized that conservation of the P.sub.1' residue is more often
important for detectable proteolysis (Vaidyanathan et al., J.
Neurochem. 72:327-337 (1999)). Thus, in particular embodiments, the
invention provides a SNAP-25 or tagged toxin substrate in which the
P.sub.1 residue is not modified or substituted relative to the
naturally occurring residue in a target protein cleaved by the
clostridial toxin. In further embodiments, the invention provides a
SNAP-25 or tagged toxin substrate in which the P.sub.1 residue is
modified or substituted relative to the naturally occurring residue
in a target protein cleaved by the clostridial toxin; such a
substrate retains susceptibility to peptide bond cleavage between
the P.sub.1 and P.sub.1' residues.
[0076] SNAP-25, VAMP and syntaxin share a short motif located
within regions predicted to adopt an .alpha.-helical conformation
(see FIG. 4). This motif is present in SNAP-25, VAMP and syntaxin
isoforms expressed in animals sensitive to the neurotoxins. In
contrast, Drosophila and yeast homologs that are resistant to these
neurotoxins and syntaxin isoforms not involved in exocytosis
contain sequence variations in the .alpha.-helical motif regions of
these VAMP and syntaxin proteins.
TABLE-US-00001 TABLE 1 Bond cleaved in human VAMP-2, SNAP-25 or
syntaxin Toxin Target
P.sub.4-P.sub.3-P.sub.2-P.sub.1-P.sub.1'-P.sub.2'-P.sub.3'-P.sub.4'
SEQ ID NO: BoNT/A SNAP-25 Glu-Ala-Asn-Gln-Arg*- SEQ ID NO: 1
Ala-Thr-Lys BoNT/B VAMP-2 Gly-Ala-Ser-Gln-Phe*- SEQ ID NO: 3
Glu-Thr-Ser BoNT/C1 syntaxin Asp-Thr-Lys-Lys-Ala*- SEQ ID NO: 5
Val-Lys-Tyr BoNT/D VAMP-2 Arg-Asp-Gln-Lys-Leu*- SEQ ID NO: 6
Ser-Glu-Leu BoNT/E SNAP-25 Gln-Ile-Asp-Arg-Ile*- SEQ ID NO: 8
Met-Glu-Lys BoNT/F VAMP-2 Glu-Arg-Asp-Gln-Lys*- SEQ ID NO: 9
Leu-Ser-Glu BoNT/G VAMP-2 Glu-Thr-Ser-Ala-Ala*- SEQ ID NO: 10
Lys-Leu-Lys TeNT VAMP-2 Gly-Ala-Ser-Gln-Phe*- SEQ ID NO: 11
Glu-Thr-Ser *Scissile bond shown in bold
[0077] Multiple repetitions of the .alpha.-helical motif are
present in proteins sensitive to cleavage by clostridial toxins:
four copies are naturally present in SNAP-25; two copies are
naturally present in VAMP; and two copies are naturally present in
syntaxin (see FIG. 4A). Furthermore, peptides corresponding to the
specific sequence of the .alpha.-helical motifs can inhibit
neurotoxin activity in vitro and in vivo, and such peptides can
cross-inhibit different neurotoxins. In addition, antibodies raised
against such peptides can cross-react among the three target
proteins, indicating that the .alpha.-helical motif is exposed on
the protein surface and adopts a similar configuration in each of
the three target proteins. Consistent with these findings,
SNAP-25-specific, VAMP-specific and syntaxin-specific neurotoxins
cross-inhibit each other by competing for the same binding site,
although they do not cleave targets non-specifically. These results
indicate that a clostridial toxin recognition sequence can include,
if desired, at least one .alpha.-helical motif. However, it is
recognized that an .alpha.-helical motif is not absolutely required
for cleavage by a clostridial toxin as evidenced by 16-mer and
17-mer peptides which serve as substrates for BoNT/A although they
lack an .alpha.-helical motif.
[0078] In one embodiment, the invention provides a SNAP-25 or
tagged toxin substrate in which the clostridial toxin recognition
sequence includes a single .alpha.-helical motif. In another
embodiment, the invention provides a SNAP-25 or tagged toxin
substrate in which the clostridial toxin recognition sequence
includes two or more .alpha.-helical motifs. As non-limiting
examples, a BoNT/A or BoNT/E recognition sequence can include a S4
.alpha.-helical motif, alone or combined with one or more
additional .alpha.-helical motifs; a BoNT/B, BoNT/G or TeNT
recognition sequence can include the V2 .alpha.-helical motif,
alone or combined with one or more additional .alpha.-helical
motifs; a BoNT/C1 recognition sequence can include the S4
.alpha.-helical motif, alone or combined with one or more
additional .alpha.-helical motifs, or an X2 .alpha.-helical motif,
alone or combined with one or more additional .alpha.-helical
motifs; and a BoNT/D or BoNT/F recognition sequence can include the
V1 .alpha.-helical motif, alone or combined with one or more
additional .alpha.-helical motifs (see FIG. 4A).
[0079] As used herein, the term "botulinum toxin serotype A
recognition sequence" is synonymous with "BoNT/A recognition
sequence" and means a scissile bond together with adjacent or
non-adjacent recognition elements, or both, sufficient for
detectable proteolysis at the scissile bond by a BoNT/A under
conditions suitable for clostridial toxin protease activity. A
scissile bond cleaved by BoNT/A can be, for example, Gln-Ala.
[0080] A variety of BoNT/A recognition sequences are well known in
the art. A BoNT/A recognition sequence can have, for example,
residues 134 to 206 or residues 137 to 206 of human SNAP-25 (Ekong
et al., supra, 1997; U.S. Pat. No. 5,962,637). A BoNT/A recognition
sequence also can include, without limitation, the sequence
Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO:
27), or a peptidomimetic thereof, which corresponds to residues 190
to 202 of human SNAP-25;
Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID
NO: 28), or a peptidomimetic thereof, which corresponds to residues
187 to 201 of human SNAP-25;
Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met
(SEQ ID NO: 29), or a peptidomimetic thereof, which corresponds to
residues 187 to 202 of human SNAP-25;
Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu
(SEQ ID NO: 30), or a peptidomimetic thereof, which corresponds to
residues 187 to 203 of human SNAP-25;
Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met
(SEQ ID NO: 31), or a peptidomimetic thereof, which corresponds to
residues 186 to 202 of human SNAP-25; or
Asp-Ser-Asn-Lys-Thr-Arg-Ile-Asp-Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met-Leu
(SEQ ID NO: 32), or a peptidomimetic thereof, which corresponds to
residues 186 to 203 of human SNAP-25. See, for example, Schmidt and
Bostian, J. Protein Chem. 14:703-708 (1995); Schmidt and Bostian,
supra, 1997; Schmidt et al., FEBS Letters 435:61-64 (1998); and
Schmidt and Bostian, U.S. Pat. No. 5,965,699). If desired, a
similar BoNT/A recognition sequence can be prepared from a
corresponding (homologous) segment of another BoNT/A-sensitive
SNAP-25 isoform or homolog such as, for example, murine, rat,
goldfish or zebrafish SNAP-25 or can be any of the peptides
disclosed herein or described in the art, for example, in U.S. Pat.
No. 5,965,699.
[0081] A BoNT/A recognition sequence can correspond to a segment of
a protein that is sensitive to cleavage by botulinum toxin serotype
A, or can be substantially similar to a segment of a
BoNT/A-sensitive protein. As illustrated in Table 2, a variety of
naturally occurring proteins sensitive to cleavage by BoNT/A are
known in the art and include, for example, human, mouse and rat
SNAP-25; and goldfish SNAP-25A and SNAP-25B. Thus, a BoNT/A
recognition sequence useful in a SNAP-25 or tagged toxin substrate
of the invention can correspond, for example, to a segment of human
SNAP-25, mouse SNAP-25, rat SNAP-25, goldfish SNAP-25A or 25B, or
another naturally occurring protein sensitive to cleavage by
BoNT/A. Furthermore, comparison of native SNAP-25 amino acid
sequences cleaved by BoNT/A reveals that such sequences are not
absolutely conserved (see Table 2 and FIG. 5), indicating that a
variety of amino acid substitutions and modifications relative to a
naturally occurring BoNT/A-sensitive SNAP-25 sequence can be
tolerated in a SNAP-25 or tagged toxin substrate of the
invention
TABLE-US-00002 TABLE 2 Cleavage of SNAP-25 and related
proteins.sup.a,b,c,d Cleavage Sites BoNT/E BoNT/A BoNT/C Resistance
to Species-Isoform SEQ ID NO: Cleavage by human 174 206 none.sup.a
mouse-SNAP-25 qnrqid ri mekadsnktridean qra tkmlgsg rat 180 end
all.sup.b human-SNAP-23 qnpqik ri tdkadtnrdridian ara kklids 179
end BoNT/A & C mouse-SNAP-23 qnqqiq ki tekadtnknridian tra
kklids 174 end BoNT/A & C chicken-SNAP-25 qnrqid ri
meklipikpglmkpt svq qrcsavvk 171 end none goldfish-SNAP-25A qnrqid
ri mdmadsnktridean qra tkmlgsg 172 end none goldfish-SNAP-25B
qnrqid ri mekadsnktridean qra tkmlgsg 180 end BoNT/E.sup.c &
A.sup.d Torpedo-SNAP-25 qnaqvd ri vvkgdmnkaridean kha tkml 180 end
(?).sup.e sea urchin-SNAP-25 qnsqvg ri tskaesnegrinsad kra knilrnk
203 end BoNT/A & C C-elegans-SNAP-25 qnrqld ri hdkgsnevrvesank
rak nlitk 182 end BoNT/E & A.sup.e Drosophila-SNAP-25 qnrqid ri
nrkgesneariavan qra hqllk 181 end BoNT/A.sup.e leech-SNAP-25 qnrqvd
ri nnkmtsnqlrisdan kra skllke .sup.a= In vitro cleavage of SNAP-25
requires 1000-fold higher BoNT/C concentration than BoNT/A or /E.
.sup.b= Substitution of p182r, or k185dd (boxes) induces
susceptibility toward BoNT/E. .sup.c= Resistance to BoNT/A possibly
due to d189 or e189 substitution by v189, see box. .sup.d= Note
that Torpedo is susceptible to BoNT/A. .sup.e= Note the presence of
several non-conservative mutations around putative cleavage
sites.
[0082] A SNAP-25 or tagged toxin substrate which includes a BoNT/A
recognition sequence can have one or multiple modifications as
compared to a naturally occurring sequence that is cleaved by
BoNT/A. For example, as compared to a 17-mer corresponding to
residues 187 to 203 of human SNAP-25, substitution of Asp193 with
Asn resulted in a relative rate of proteolysis of 0.23;
substitution of Glu194 with Gln resulted in a relative rate of
2.08; substitution of Ala195 with 2-aminobutyric acid resulted in a
relative rate of 0.38; and substitution of Gln197 with Asn,
2-aminobutyric acid or Ala resulted in a relative rate of 0.66,
0.25, or 0.19, respectively (see Table 3). Furthermore,
substitution of Ala199 with 2-aminobutyric acid resulted in a
relative rate of 0.79; substitution of Thr200 with Ser or
2-aminobutyric acid resulted in a relative rate of 0.26 or 1.20,
respectively; substitution of Lys201 with Ala resulted in a
relative rate of 0.12; and substitution of Met202 with Ala or
norleucine resulted in a relative rate of 0.38 or 1.20,
respectively. See Schmidt and Bostian, supra, 1997. These results
indicate that a variety of residues can be substituted in a SNAP-25
or tagged toxin substrate as compared to a naturally occurring
toxin-sensitive sequence. In the case of BoNT/A, these results
indicate that residues including but not limited to Glu194, Ala195,
Gln197, Ala199, Thr200 and Met202, Leu203, Gly204, Ser205, and
Gly206, as well as residues more distal from the Gln-Arg scissile
bond can be substituted to produce a SNAP-25 or tagged toxin
substrate of the invention. Such a substrate is detectably
proteolyzed at the scissile bond by BoNT/A under conditions
suitable for clostridial toxin protease activity. In sum, it is
understood that a SNAP-25 or tagged toxin substrate can include, if
desired, one or several amino acid substitutions, additions or
deletions relative to a naturally occurring SNAP-25 sequence. A
SNAP-25 or tagged toxin substrate also can optionally include a
carboxy-terminal amide.
TABLE-US-00003 TABLE 3 Kinetic parameters of BoNT/A synthetic
peptide substrates Relative Peptide Sequence.sup.a SEQ ID NO:
Rate.sup.b [1-15] SNKTRIDEANQRATK 28 0.03 [1-16] SNKTRIDEANQRATKM
29 1.17 [1-17] SNKTRIDEANQRATKML 30 1.00 M16A SNKTRIDEANQRATKAL 44
0.38 M16X SNKTRIDEANQRATKXL 45 1.20 K15A SNKTRIDEANQRATAML 46 0.12
T14S SNKTRIDEANQRASKML 47 0.26 T14B SNKTRIDEANQRABKML 48 1.20 A13B
SNKTRIDEANQRBTKML 49 0.79 Q11A SNKTRIDEANARATKML 50 0.19 Q11B
SNKTRIDEANBRATKML 51 0.25 Q11N SNKTRIDEANNRATKML 52 0.66 N10A
SNKTRIDEAAQRATKML 53 0.06 A9B SNKTRIDEBNQRATKML 54 0.38 E8Q
SNKTRIDQANQRATKML 55 2.08 D7N SNKTRINEANQRATKML 56 0.23
.sup.aNonstandard amino acid abbreviations are: B, 2-aminobutyric
acid; X, 2-aminohexanoic acid (norleucine) .sup.bInitial hydrolysis
rates relative to peptide [1-17]. Peptide concentrations were 1.0
mM.
TABLE-US-00004 TABLE 4 Cleavage of VAMP.sup.a,.sup.b Cleavage Sites
BoNT/B BoNT/F BoNT/D TeNT BoNT/G Resistance to Species-Isoform SEQ
ID NO: Cleavage by human 53 92 none mouse-VAMP-1 dkvlerd qkl
selddradalqagas qf ess aa klkrkyww bovine human 51 90 none
mouse-VAMP-2 dkvlerd qkl selddradalqagas qf ets aa klkrkyww bovine
53 92 TeNT & rat-VAMP-2 dkvlerd qkl selddradalqagas vf ess aa
klkrkyww BoNT/B 51 90 none rat-VAMP-2 dkvlerd qkl selddradalqagas
qf ets aa klkrkyww 38 77 none rat-Cellubrevin dkvlerd qkl
selddradalqagas qf ets aa klkrkyww 146 175 all rat-TI-VAMP dlvaqrg
erl ellidktenlvdssv tf ktt sr nlaramcm -- -- TeNT &
chicken-VAMP-1 ----erd qkl selddradalqagas vf ess aa klkr----
BoNT/B -- -- none chicken-VAMP-2 ----erd qkl selddradalqagas qf ets
aa klkr--- 55 94 none Torpedo-VAMP-1 dkvlerd qkl selddradalqagas qf
ess aa klkrkyww 35 74 BoNT F, D sea urchin-VAMP dkvldrd qal
svlddradalqqgas qf etn ag klkrkyww & G 41 80 BoNT/G
Aplysia-VAMP ekvldrd qki sqlddraealqagas qf eas ag klkrkyww 60 99
BoNT F & squid-VAMP dkvlerd ski selddradalqagas qf eas ag
klkrkfww G 86 115 BoNT F, D C. elegans-VAMP nkvmerd vql
nsldhraevlqngas qf qqs sr elkrgyww & G 67 106 TeNT &
Drosphila-syb.sup.a ekvlerd qkl selgeradqleqgas qs eqq ag klkrkqww
BoNT B & G 61 100 BoNT/F & Drosphila-n-syb.sup.b ekvlerd
skl selddradalqqgas qf eqq ag klkrkfwl G 49 88 BoNT/G leech-VAMP
dkvlekd qkl aeldgradalqagas qf eas ag klkrkfww .sup.a= Sequence
corrected in position 93 (f > s). .sup.b= Sequence corrected in
position 68 (t > s).
TABLE-US-00005 TABLE 5 Cleavage of syntaxin Cleavage Sites BoNT/C
Resistance to Species-Isoform SEQ ID NO: Cleavage by human 245 262
no rat-syntaxin 1A eravsdtk ka vkyqskar mouse bovine human 244 261
no rat-syntaxin 1B eravsdtk ka vkyqskar mouse bovine 245 262 no
rat-syntaxin 2 ehakeetk ka ikyqskar 244 261 no rat-syntaxin 3
ekardetr ka mkyqgqar 244 261 yes rat-syntaxin 4 ergqehvk ia
lenqkkar 239 259 expected chicken-syntaxin 1B vpevfvtk sa vmyqcksr
243 260 no sea urchin-syntaxin vrrqndtk ka vkyqskar 247 264 no
Aplysia-syntaxin 1 etakmdtk ka vkyqskar 248 265 no squid-syntaxin
etakvdtk ka vkyqskar 248 265 no Drosophila-Dsynt 1 qtatqdtk ka
lkyqskar 251 268 no leech-syntaxin 1 etaaadtk ka mkyqsaar
[0083] As used herein, the term "botulinum toxin serotype B
recognition sequence" is synonymous with "BoNT/B recognition
sequence" and means a scissile bond together with adjacent or
non-adjacent recognition elements, or both, sufficient for
detectable proteolysis at the scissile bond by a BoNT/B under
appropriate conditions. A scissile bond cleaved by BoNT/B can be,
for example, Gln-Phe.
[0084] A variety of BoNT/B recognition sequences are well known in
the art or can be defined by routine methods. Such a BoNT/B
recognition sequence can include, for example, a sequence
corresponding to some or all of the hydrophilic core of a VAMP
protein such as human VAMP-1 or human VAMP-2. A BoNT/B recognition
sequence can include, without limitation, residues 33 to 94,
residues 45 to 94, residues 55 to 94, residues 60 to 94, residues
65 to 94, residues 60 to 88 or residues 65 to 88 of human VAMP-2
(SEQ ID NO: 4), or residues 60 to 94 of human VAMP-1 (SEQ ID NO:
96) (see, for example, Shone et al., Eur. J. Biochem. 217: 965-971
(1993) and U.S. Pat. No. 5,962,637). If desired, a similar BoNT/B
recognition sequence can be prepared from a corresponding
(homologous) segment of another BoNT/B-sensitive VAMP isoform or
homolog such as human VAMP-1 or rat or chicken VAMP-2.
[0085] Thus, it is understood that a BoNT/B recognition sequence
can correspond to a segment of a protein that is sensitive to
cleavage by botulinum toxin serotype B, or can be substantially
similar to such a segment of a BoNT/B-sensitive protein. As shown
in Table 4, a variety of naturally occurring proteins sensitive to
cleavage by BoNT/B are known in the art and include, for example,
human, mouse and bovine VAMP-1 and VAMP-2; rat VAMP-2; rat
cellubrevin; chicken VAMP-2; Torpedo VAMP-1; sea urchin VAMP;
Aplysia VAMP; squid VAMP; C. elegans VAMP; Drosophila n-syb; and
leech VAMP. Thus, a BoNT/B recognition sequence useful in a tagged
toxin substrate of the invention can correspond, for example, to a
segment of human VAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine
VAMP-1 or VAMP-2, rat VAMP-2, rat cellubrevin, chicken VAMP-2,
Torpedo VAMP-1, sea urchin VAMP, Aplysia VAMP, squid VAMP, C.
elegans VAMP, Drosophila n-syb, leech VAMP, or another naturally
occurring protein sensitive to cleavage by BoNT/B. Furthermore, as
shown in Table 4, comparison of native VAMP amino acid sequences
cleaved by BoNT/B reveals that such sequences are not absolutely
conserved (see, also, FIG. 6), indicating that a variety of amino
acid substitutions and modifications relative to a naturally
occurring VAMP sequence can be tolerated in a tagged toxin
substrate which includes a BoNT/A recognition sequence.
[0086] As used herein, the term "botulinum toxin serotype C1
recognition sequence" is synonymous with "BoNT/C1 recognition
sequence" and means a scissile bond together with adjacent or
non-adjacent recognition elements, or both, sufficient for
detectable proteolysis at the scissile bond by a BoNT/C1 under
appropriate conditions. A scissile bond cleaved by BoNT/C1 can be,
for example, Lys-Ala or Arg-Ala.
[0087] It is understood that a BoNT/C1 recognition sequence can
correspond to a segment of a protein that is sensitive to cleavage
by botulinum toxin serotype C1, or can be substantially similar to
a segment of a BoNT/C1-sensitive protein. As shown in Table 5, a
variety of naturally occurring proteins sensitive to cleavage by
BoNT/C1 are known in the art and include, for example, human, rat,
mouse and bovine syntaxin 1A and 1B; rat syntaxins 2 and 3; sea
urchin syntaxin; Aplysia syntaxin 1; squid syntaxin; Drosophila
Dsynt1; and leech syntaxin 1. Thus, a BoNT/C1 recognition sequence
useful in a tagged toxin substrate of the invention can correspond,
for example, to a segment of human, rat, mouse or bovine syntaxin
1A or 1B, rat syntaxin 2, rat syntaxin 3, sea urchin syntaxin,
Aplysia syntaxin 1, squid syntaxin, Drosophila Dsynt1, leech
syntaxin 1, or another naturally occurring protein sensitive to
cleavage by BoNT/C1. Furthermore, comparison of native syntaxin
amino acid sequences cleaved by BoNT/C1 reveals that such sequences
are not absolutely conserved (see Table 5 and FIG. 7), indicating
that a variety of amino acid substitutions and modifications
relative to a naturally occurring BoNT/C1-sensitive syntaxin
sequence can be tolerated in a tagged toxin substrate including a
BoNT/C1 recognition sequence.
[0088] A variety of naturally occurring SNAP-25 proteins also are
sensitive to cleavage by BoNT/C1, including human, mouse and rat
SNAP-25; goldfish SNAP-25A and 25B; and Drosophila and leech
SNAP-25. Thus, a BoNT/C1 recognition sequence useful in a SNAP-25
or tagged toxin substrate of the invention can correspond, for
example, to a segment of human, mouse or rat SNAP-25, goldfish
SNAP-25A or 25B, Torpedo SNAP-25, zebrafish SNAP-25, Drosophila
SNAP-25, leech SNAP-25, or another naturally occurring protein
sensitive to cleavage by BoNT/C1. As discussed above in regard to
variants of naturally occurring syntaxin sequences, comparison of
native SNAP-25 amino acid sequences cleaved by BoNT/C1 reveals
significant sequence variability (see Table 2 and FIG. 5 above),
indicating that a variety of amino acid substitutions and
modifications relative to a naturally occurring BoNT/C1-sensitive
SNAP-25 sequence can be tolerated in a SNAP-25 or tagged toxin
substrate of the invention.
[0089] The term "botulinum toxin serotype D recognition sequence"
is synonymous with "BoNT/D recognition sequence" and means a
scissile bond together with adjacent or non-adjacent recognition
elements, or both, sufficient for detectable proteolysis at the
scissile bond by a BoNT/D under appropriate conditions. A scissile
bond cleaved by BoNT/D can be, for example, Lys-Leu.
[0090] A variety of BoNT/D recognition sequences are well known in
the art or can be defined by routine methods. A BoNT/D recognition
sequence can include, for example, residues 27 to 116; residues 37
to 116; residues 1 to 86; residues 1 to 76; or residues 1 to 69 of
rat VAMP-2 (SEQ ID NO: 7; Yamasaki et al., J. Biol. Chem.
269:12764-12772 (1994)). Thus, a BoNT/D recognition sequence can
include, for example, residues 27 to 69 or residues 37 to 69 of rat
VAMP-2 (SEQ ID NO: 7). If desired, a similar BoNT/D recognition
sequence can be prepared from a corresponding (homologous) segment
of another BoNT/D-sensitive VAMP isoform or homolog such as human
VAMP-1 or human VAMP-2.
[0091] A BoNT/D recognition sequence can correspond to a segment of
a protein that is sensitive to cleavage by botulinum toxin serotype
D, or can be substantially similar to a segment of a
BoNT/D-sensitive protein. As shown in Table 5, a variety of
naturally occurring proteins sensitive to cleavage by BoNT/D are
known in the art and include, for example, human, mouse and bovine
VAMP-1 and VAMP-2; rat VAMP-1 and VAMP-2; rat cellubrevin; chicken
VAMP-1 and VAMP-2; Torpedo VAMP-1; Aplysia VAMP; squid VAMP;
Drosophila syb and n-syb; and leech VAMP. Thus, a BoNT/D
recognition sequence useful in a tagged toxin substrate of the
invention can correspond, for example, to a segment of human VAMP-1
or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat
VAMP-1 or VAMP-2, rat cellubrevin, chicken VAMP-1 or VAMP-2,
Torpedo VAMP-1, Aplysia VAMP, squid VAMP, Drosophila syb or n-syb,
leech VAMP, or another naturally occurring protein sensitive to
cleavage by BoNT/D. Furthermore, as shown in Table 5 above,
comparison of native VAMP amino acid sequences cleaved by BoNT/D
reveals significant sequence variability (see, also, FIG. 6),
indicating that a variety of amino acid substitutions and
modifications relative to a naturally occurring BoNT/D-sensitive
VAMP sequence can be tolerated in a tagged toxin substrate of the
invention.
[0092] As used herein, the term "botulinum toxin serotype E
recognition sequence" is synonymous with "BoNT/E recognition
sequence" and means a scissile bond together with adjacent or
non-adjacent recognition elements, or both, sufficient for
detectable proteolysis at the scissile bond by a BoNT/E under
appropriate conditions. A scissile bond cleaved by BoNT/E can be,
for example, Arg-Ile.
[0093] One skilled in the art appreciates that a BoNT/E recognition
sequence can correspond to a segment of a protein that is sensitive
to cleavage by botulinum toxin serotype E, or can be substantially
similar to a segment of a BoNT/E-sensitive protein. A variety of
naturally occurring proteins sensitive to cleavage by BoNT/E are
known in the art and include, for example, human, mouse and rat
SNAP-25; mouse SNAP-23; chicken SNAP-25; goldfish SNAP-25A and
SNAP-25B; zebrafish SNAP-25; C. elegans SNAP-25; and leech SNAP-25
(see Table 2). Thus, a BoNT/E recognition sequence useful in a
SNAP-25 or tagged toxin substrate of the invention can correspond,
for example, to a segment of human SNAP-25, mouse SNAP-25, rat
SNAP-25, mouse SNAP-23, chicken SNAP-25, goldfish SNAP-25A or 25B,
C. elegans SNAP-25, leech SNAP-25, or another naturally occurring
protein sensitive to cleavage by BoNT/E. Furthermore, as shown in
Table 2 and FIG. 5 above, comparison of native SNAP-23 and SNAP-25
amino acid sequences cleaved by BoNT/E reveals that such sequences
are not absolutely conserved, indicating that a variety of amino
acid substitutions and modifications relative to a naturally
occurring BoNT/E-sensitive SNAP-23 or SNAP-25 sequence can be
tolerated in a SNAP-25 or tagged toxin substrate of the
invention.
[0094] The term "botulinum toxin serotype F recognition sequence,"
as used herein, is synonymous with "BoNT/F recognition sequence"
and means a scissile bond together with adjacent or non-adjacent
recognition elements, or both, sufficient for detectable
proteolysis at the scissile bond by a BoNT/F under appropriate
conditions. A scissile bond cleaved by BoNT/F can be, for example,
Gln-Lys.
[0095] A variety of BoNT/F recognition sequences are well known in
the art or can be defined by routine methods. A BoNT/F recognition
sequence can include, for example, residues 27 to 116; residues 37
to 116; residues 1 to 86; residues 1 to 76; or residues 1 to 69 of
rat VAMP-2 ((SEQ ID NO: 7; Yamasaki et al., supra, 1994). A BoNT/F
recognition sequence also can include, for example, residues 27 to
69 or residues 37 to 69 of rat VAMP-2 (SEQ ID NO: 7). It is
understood that a similar BoNT/F recognition sequence can be
prepared, if desired, from a corresponding (homologous) segment of
another BoNT/F-sensitive VAMP isoform or homolog such as human
VAMP-1 or human VAMP-2.
[0096] A BoNT/F recognition sequence can correspond to a segment of
a protein that is sensitive to cleavage by botulinum toxin serotype
F, or can be substantially similar to a segment of a
BoNT/F-sensitive protein. A variety of naturally occurring proteins
sensitive to cleavage by BoNT/F are known in the art and include,
for example, human, mouse and bovine VAMP-1 and VAMP-2; rat VAMP-1
and VAMP-2; rat cellubrevin; chicken VAMP-1 and VAMP-2; Torpedo
VAMP-1; Aplysia VAMP; Drosophila syb; and leech VAMP (see Table 5).
Thus, a BoNT/F recognition sequence useful in a tagged toxin
substrate of the invention can correspond, for example, to a
segment of human VAMP-1 or VAMP-2, mouse VAMP-1 or VAMP-2, bovine
VAMP-1 or VAMP-2, rat VAMP-1 or VAMP-2, rat cellubrevin, chicken
VAMP-1 or VAMP-2, Torpedo VAMP-1, Aplysia VAMP, Drosophila syb,
leech VAMP, or another naturally occurring protein sensitive to
cleavage by BoNT/F. Furthermore, as shown in Table 5 above,
comparison of native VAMP amino acid sequences cleaved by BoNT/F
reveals that such sequences are not absolutely conserved (see,
also, FIG. 6), indicating that a variety of amino acid
substitutions and modifications relative to a naturally occurring
BoNT/F-sensitive VAMP sequence can be tolerated in a tagged toxin
substrate which includes a BoNT/F recognition sequence.
[0097] As used herein, the term "botulinum toxin serotype G
recognition sequence" is synonymous with "BoNT/G recognition
sequence" and means a scissile bond together with adjacent or
non-adjacent recognition elements, or both, sufficient for
detectable proteolysis at the scissile bond by a BoNT/G under
appropriate conditions. A scissile bond cleaved by BoNT/G can be,
for example, Ala-Ala.
[0098] A BoNT/G recognition sequence can correspond to a segment of
a protein that is sensitive to cleavage by botulinum toxin serotype
G, or can be substantially similar to such a BoNT/G-sensitive
segment. As illustrated in Table 5 above, a variety of naturally
occurring proteins sensitive to cleavage by BoNT/G are known in the
art and include, for example, human, mouse and bovine VAMP-1 and
VAMP-2; rat VAMP-1 and VAMP-2; rat cellubrevin; chicken VAMP-1 and
VAMP-2; and Torpedo VAMP-1. Thus, a BoNT/G recognition sequence
useful in a tagged toxin substrate of the invention can correspond,
for example, to a segment of human VAMP-1 or VAMP-2, mouse VAMP-1
or VAMP-2, bovine VAMP-1 or VAMP-2, rat VAMP-1 or VAMP-2, rat
cellubrevin, chicken VAMP-1 or VAMP-2, Torpedo VAMP-1, or another
naturally occurring protein sensitive to cleavage by BoNT/G.
Furthermore, as shown in Table 5 above, comparison of native VAMP
amino acid sequences cleaved by BoNT/G reveals that such sequences
are not absolutely conserved (see, also, FIG. 6), indicating that a
variety of amino acid substitutions and modifications relative to a
naturally occurring BoNT/G-sensitive VAMP sequence can be tolerated
in a tagged toxin substrate which includes a BoNT/G recognition
sequence.
[0099] The term "tetanus toxin recognition sequence" means a
scissile bond together with adjacent or non-adjacent recognition
elements, or both, sufficient for detectable proteolysis at the
scissile bond by a tetanus toxin under appropriate conditions. A
scissile bond cleaved by TeNT can be, for example, Gln-Phe.
[0100] A variety of TeNT recognition sequences are well known in
the art or can be defined by routine methods and include a sequence
corresponding to some or all of the hydrophilic core of a VAMP
protein such as human VAMP-1 or human VAMP-2. A TeNT recognition
sequence can include, for example, residues 25 to 93 or residues 33
to 94 of human VAMP-2 (SEQ ID NO: 4; Cornille et al., Eur. J.
Biochem. 222:173-181 (1994); Foran et al., Biochem. 33: 15365-15374
(1994)); residues 51 to 93 or residues 1 to 86 of rat VAMP-2 (SEQ
ID NO: 7; Yamasaki et al., supra, 1994); or residues 33 to 94 of
human VAMP-1 (SEQ ID NO: 96). A TeNT recognition sequence also can
include, for example, residues 25 to 86, residues 33 to 86 or
residues 51 to 86 of human VAMP-2 (SEQ ID NO: 4) or rat VAMP-2 (SEQ
ID NO: 7). It is understood that a similar TeNT recognition
sequence can be prepared, if desired, from a corresponding
(homologous) segment of another TeNT-sensitive VAMP isoform or
species homolog such as human VAMP-1 or sea urchin or Aplysia
VAMP.
[0101] Thus, a TeNT recognition sequence can correspond to a
segment of a protein that is sensitive to cleavage by tetanus
toxin, or can be substantially similar to a segment of a
TeNT-sensitive protein. As shown in Table 5 above, a variety of
naturally occurring proteins sensitive to cleavage by TeNT are
known in the art and include, for example, human, mouse and bovine
VAMP-1 and VAMP-2; rat VAMP-2; rat cellubrevin; chicken VAMP-2;
Torpedo VAMP-1; sea urchin VAMP; Aplysia VAMP; squid VAMP; C.
elegans VAMP; Drosophila n-syb; and leech VAMP. Thus, a TeNT
recognition sequence useful in a tagged toxin substrate of the
invention can correspond, for example, to a segment of human VAMP-1
or VAMP-2, mouse VAMP-1 or VAMP-2, bovine VAMP-1 or VAMP-2, rat
VAMP-2, rat cellubrevin, chicken VAMP-2, Torpedo VAMP-1, sea urchin
VAMP, Aplysia VAMP, squid VAMP, C. elegans VAMP, Drosophila n-syb,
leech VAMP, or another naturally occurring protein sensitive to
cleavage by TeNT. Furthermore, comparison of native VAMP amino acid
sequences cleaved by TeNT reveals that such sequences are not
absolutely conserved (Table 5 and FIG. 6), indicating that a
variety of amino acid substitutions and modifications relative to a
naturally occurring TeNT-sensitive VAMP sequence can be tolerated
in a tagged toxin substrate which includes a TeNT recognition
sequence.
[0102] In view of the above, it is clear that a "portion of
SNAP-25" included in a SNAP-25 substrate, or a "clostridial toxin
recognition sequence" included in a tagged toxin substrate, can
correspond to a segment of SNAP-25, VAMP or syntaxin which is less
than full-length SNAP-25, VAMP or syntaxin. In particular
embodiments, a BoNT/A recognition sequence is homologous to at most
160, 140, 120, 100, 80, 60, 40, 20 or 10 consecutive residues of
SNAP-25, where the consecutive residues include the cleavage site
Gln-Arg. As non-limiting examples, a BoNT/A recognition sequence
can have at least 80% amino acid identity with at most 160, 140,
120, 100, 80, 60, 40, 20 or 10 consecutive residues of human
SNAP-25 (SEQ ID NO: 2) or another SNAP-25, where the consecutive
residues include the cleavage site Gln-Arg.
[0103] In other embodiments, a BoNT/B recognition sequence is
homologous to at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of VAMP, where the consecutive residues
include the cleavage site Gln-Phe. As non-limiting examples, a
BoNT/B recognition sequence can have at least 80% amino acid
identity with at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of human VAMP-1 (SEQ ID NO: 96) or human
VAMP-2 (SEQ ID NO: 4) or another VAMP, where the consecutive
residues include the cleavage site Gln-Phe.
[0104] In further embodiments, a BoNT/C1 recognition sequence is
homologous to at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of syntaxin, where the consecutive residues
include the cleavage site Lys-Ala. As non-limiting examples, a
BoNT/C1 recognition sequence can have at least 80% amino acid
identity with at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of human syntaxin 1A (SEQ ID NO: 21) or human
syntaxin-1B or another syntaxin, where the consecutive residues
include the cleavage site Lys-Ala.
[0105] In still further embodiments, a BoNT/C1 recognition sequence
is homologous to at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of SNAP-25, where the consecutive residues
include the cleavage site Arg-Ala. As non-limiting examples, a
BoNT/C1 recognition sequence can have at least 80% amino acid
identity with at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of human SNAP-25 (SEQ ID NO: 2) or another
SNAP-25, where the consecutive residues include the cleavage site
Arg-Ala.
[0106] In additional embodiments, a BoNT/D recognition sequence is
homologous to at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of VAMP, where the consecutive residues
include the cleavage site Lys-Leu. As non-limiting examples, a
BoNT/D recognition sequence can have at least 80% amino acid
identity with at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of human VAMP-1 (SEQ ID NO: 96) or human
VAMP-2 (SEQ ID NO: 4) or another VAMP, where the consecutive
residues include the cleavage site Lys-Leu.
[0107] In other embodiments, a BoNT/E recognition sequence is
homologous to at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of SNAP-25, where the consecutive residues
include the cleavage site Arg-Ile. As non-limiting examples, a
BoNT/E recognition sequence can have at least 80% amino acid
identity with at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of human SNAP-25 (SEQ ID NO: 2) or another
SNAP-25, where the consecutive residues include the cleavage site
Arg-Ile.
[0108] In further embodiments, a BoNT/F recognition sequence is
homologous to at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of VAMP, where the consecutive residues
include the cleavage site Gln-Lys. As non-limiting examples, a
BoNT/F recognition sequence can have at least 80% amino acid
identity with at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of human VAMP-1 (SEQ ID NO: 96) or human
VAMP-2 (SEQ ID NO: 4) or another VAMP, where the consecutive
residues include the cleavage site Gln-Lys.
[0109] In yet further embodiments, a BoNT/G recognition sequence is
homologous to at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of VAMP, where the consecutive residues
include the cleavage site Ala-Ala. As non-limiting examples, a
BoNT/G recognition sequence can have at least 80% amino acid
identity with at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of human VAMP-1 (SEQ ID NO: 96) or human
VAMP-2 (SEQ ID NO: 4) or another VAMP, where the consecutive
residues include the cleavage site Ala-Ala.
[0110] In still further embodiments, a TeNT recognition sequence is
homologous to at most 160, 140, 120, 100, 80, 60, 40, 20 or 10
consecutive residues of VAMP, where the consecutive residues
include the cleavage site Gln-Phe. As non-limiting examples, a TeNT
recognition sequence can have at least 80% amino acid identity with
at most 160, 140, 120, 100, 80, 60, 40, 20 or 10 consecutive
residues of human VAMP-1 (SEQ ID NO: 96) or human VAMP-2 (SEQ ID
NO: 4) or another VAMP, where the consecutive residues include the
cleavage site Gln-Phe.
[0111] In another embodiment, a clostridial toxin recognition
sequence is a sequence other than a substrate sequence described in
U.S. Pat. No. 7,762,280. In further embodiments, a clostridial
toxin recognition sequence is a sequence other than
SNRTRIDEANQRATRMLG (SEQ ID NO: 109); LSELDDRADALQAGASQ
FETSAAKLKRKYWWKNLK (SEQ ID NO: 110); AQVDEVVDIMRVNVDKVLER
DQKLSELDDRADALQAGAS (SEQ ID NO: 111);
NKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENL
EQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG (SEQ ID
NO: 112); or
NKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGI-
IGNLRH M ALDMGNEIDTQNRQIDRIMEKADSNKTRI DEANQAATKMLGSG (SEQ ID NO:
113).
[0112] A SNAP-25 or tagged toxin substrate also can contain one or
multiple clostridial toxin cleavage sites for the same or different
clostridial toxin. In one embodiment, a SNAP-25 or tagged toxin
substrate contains a single cleavage site. In another embodiment, a
SNAP-25 or tagged toxin substrate has multiple cleavage sites for
the same clostridial toxin. These cleavage sites can be
incorporated within the same or different clostridial toxin
recognition sequences. In a further embodiment, a SNAP-25 or tagged
toxin substrate has multiple cleavage sites for the same
clostridial toxin that intervene between the same green fluorescent
protein, or other fluorescent protein or genetically encoded
detectable marker, and the first partner of the affinity couple. A
SNAP-25 or tagged toxin substrate can contain, for example, two or
more, three or more, five or more, seven or more, eight or more, or
ten or more cleavage sites for the same clostridial toxin
intervening between the same or different green fluorescent
protein, or other fluorescent protein or genetically encoded
detectable marker, and the first partner of the affinity couple. A
SNAP-25 or tagged toxin substrate also can have, for example, two,
three, four, five, six, seven, eight, nine or ten cleavage sites
for the same clostridial toxin intervening between the same or
different green fluorescent protein, or other fluorescent protein
or genetically encoded detectable marker, and the first partner of
the affinity couple.
[0113] A SNAP-25 or tagged toxin substrate also can contain
multiple cleavage sites for different clostridial toxins. In one
embodiment, a SNAP-25 or tagged toxin substrate includes multiple
cleavage sites for different clostridial toxins all intervening
between the same green fluorescent protein, or other fluorescent
protein or genetically encoded detectable marker, and first partner
of the affinity couple. A SNAP-25 or tagged toxin substrate can
contain, for example, two or more, three or more, five or more, or
ten or more cleavage sites for different clostridial toxins all
intervening between the same green fluorescent protein, or other
fluorescent protein or genetically encoded detectable marker, and
first partner of the affinity couple. A SNAP-25 or tagged toxin
substrate also can contain, for example, two or more, three or
more, five or more, or ten or more cleavage sites for different
clostridial toxins intervening between at least two different pairs
of green fluorescent proteins, or other fluorescent proteins or
genetically encoded detectable markers, and first partners of an
affinity couple. In particular embodiments, a clostridial substrate
also has two, three, four, five, six, seven, eight, nine or ten
cleavage sites for different clostridial toxins, where the cleavage
sites intervene between the same or different pairs of green
fluorescent proteins, or other fluorescent proteins or genetically
encoded detectable markers, and first partners of an affinity
couple. It is understood that a SNAP-25 or tagged toxin substrate
having multiple cleavage sites can have any combination of two,
three, four, five, six, seven or eight cleavage sites for any
combination of the following clostridial toxins: BoNT/A, BoNT/B,
BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT.
[0114] It is understood that, in addition to a green fluorescent
protein, or other fluorescent protein or genetically encoded
detectable marker, a first partner of an affinity couple, and a
clostridial toxin recognition sequence, a SNAP-25 substrate or
tagged toxin substrate can include, if desired, one or more
additional components. As an example, a flexible spacer sequence
such as GGGGS (SEQ ID NO: 84) can be included in a SNAP-25 or
tagged toxin substrate of the invention. A SNAP-25 or tagged toxin
substrate further can include, without limitation, one or more of
the following: a carboxy-terminal cysteine residue; an
immunoglobulin hinge region; an N-hydroxysuccinimide linker; a
peptide or peptidomimetic hairpin turn; a hydrophilic sequence, or
another component or sequence that promotes the solubility or
stability of the SNAP-25 or tagged toxin substrate.
[0115] Furthermore, a SNAP-25 or tagged toxin substrate can be
cleaved at a reduced or enhanced rate relative to SNAP-25, VAMP or
syntaxin proteins or relative to a similar peptide or
peptidomimetic that does not contain a fluorescent protein or
genetically encoded detectable marker or a first partner of an
affinity couple.
[0116] A SNAP-25 or tagged toxin substrate such as a BoNT/A,
BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G or TeNT substrate
can be cleaved, for example, with an initial hydrolysis rate that
is at least 5% of the initial hydrolysis rate, under otherwise
identical conditions, of human SNAP-25, VAMP or syntaxin, where the
SNAP-25 or tagged toxin substrate and SNAP-25, VAMP or syntaxin
each is present at a concentration of 16 .mu.M.
[0117] Where a SNAP-25 or tagged toxin substrate includes a BoNT/A,
BoNT/C1 or BoNT/E recognition sequence, the substrate can be
cleaved, for example, with an initial hydrolysis rate that is at
least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,
200%, 250%, or 300% of the initial hydrolysis rate, under otherwise
identical conditions, of human SNAP-25 by BoNT/A, BoNT/C1 or
BoNT/E, respectively, where the substrate and human SNAP-25 each is
present at a concentration of 16 .mu.M. In other embodiments, such
a substrate is cleaved with an initial hydrolysis rate that is at
least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,
200%, 250%, or 300% of the initial hydrolysis rate, under otherwise
identical conditions, of human SNAP-25 by BoNT/A, BoNT/C1 or
BoNT/E, respectively, where the SNAP-25 or tagged toxin substrate
and human SNAP-25 each is present at a concentration of 200
.mu.M.
[0118] Similarly, where a SNAP-25 or tagged toxin substrate
includes a BoNT/B, BoNT/D, BoNT/F or BoNT/G recognition sequence,
the substrate can be cleaved, for example, with an initial
hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initial
hydrolysis rate, under otherwise identical conditions, of human
VAMP-2 by BoNT/B, BoNT/D, BoNT/F or BoNT/G, respectively, where
substrate of the invention and human VAMP-2 each is present at a
concentration of 16 .mu.M. In other embodiments, such a substrate
is cleaved with an initial hydrolysis rate that is at least 5%,
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,
250%, or 300% of the initial hydrolysis rate, under otherwise
identical conditions, of human VAMP-2 by BoNT/B, BoNT/D, BoNT/F or
BoNT/G, respectively, where the SNAP-25 or tagged toxin substrate
and human VAMP-2 each is present at a concentration of 200
.mu.M.
[0119] Where a tagged toxin substrate includes a BoNT/C1
recognition sequence, the substrate can be cleaved with an initial
hydrolysis rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% of the initial
hydrolysis rate, under otherwise identical conditions, of human
syntaxin by BoNT/C1, where the tagged toxin substrate and human
syntaxin each is present at a concentration of 16 .mu.M. In other
embodiments, such a substrate is cleaved with an initial hydrolysis
rate that is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 100%, 150%, 200%, 250%, or 300% of the initial hydrolysis
rate, under otherwise identical conditions, of human syntaxin by
BoNT/C1, where the tagged toxin substrate and human syntaxin each
is present at a concentration of 200 .mu.M.
[0120] The "turnover number," or k.sub.cat, is the rate of
breakdown of a toxin-substrate complex. A SNAP-25 or tagged toxin
substrate can be cleaved with a k.sub.cat that is reduced or
enhanced as compared to the k.sub.cat of human SNAP-25, human
VAMP-2 or human syntaxin target proteins when cleaved by the same
clostridial toxin under the same conditions. A SNAP-25 or tagged
toxin substrate can be cleaved, for example, with a k.sub.cat of
about 0.001 to about 4000 sec.sup.-1. In one embodiment, a SNAP-25
or tagged toxin substrate is cleaved with a k.sub.cat of about 1 to
about 4000 sec.sup.-1. In other embodiments, a SNAP-25 or tagged
toxin substrate has a k.sub.cat of less than 5 sec.sup.-1, 10
sec.sup.1, 25 sec.sup.-1, 50 sec.sup.-1, 100 sec.sup.1, 250
sec.sup.1, 500 sec.sup.-1, or 1000 sec.sup.-1. A SNAP-25 or tagged
toxin substrate also can have, for example, a k.sub.cat in the
range of 1 to 1000 sec.sup.-1; 1 to 500 sec.sup.-; 1 to 250
sec.sup.-1; 1 to 100 sec.sup.1; 1 to 50 sec.sup.-1; 10 to 1000
sec.sup.-1; 10 to 500 sec.sup.-1; 10 to 250 sec.sup.-1; 0 to 100
sec.sup.-1; 10 to 50 sec.sup.-1; 25 to 1000 sec.sup.-1; 25 to 500
sec.sup.-1; 25 to 250 sec.sup.-1; 25 to 100 sec.sup.-1; 25 to 50
sec.sup.-1; 50 to 1000 sec.sup.-; 50 to 500 sec.sup.-1; 50 to 250
sec.sup.-1; 50 to 100 sec.sup.-; 100 to 1000 sec.sup.1; 100 to 500
sec.sup.-1; or 100 to 250 sec.sup.-1. One skilled in the art
understands the turnover number, k.sub.cat, is assayed under
standard steady state conditions in which there is an excess of
substrate.
[0121] One skilled in the art understands that there are several
considerations in selecting and positioning a green fluorescent
protein, or other fluorescent protein or genetically encoded
detectable marker and a first partner of an affinity couple in a
SNAP-25 or tagged toxin substrate. These elements generally are
positioned to minimize interference with substrate binding to, or
proteolysis by, the clostridial toxin. Thus, a green fluorescent
protein, or other fluorescent protein or genetically encoded
detectable marker, and a first partner of an affinity couple can be
selected and positioned, for example, so as to minimize the
disruption of bonded and non-bonded interactions that are important
for binding, and to minimize steric hindrance.
[0122] In a complex of a VAMP substrate and the light chain of
BoNT/B (LC/B), nearly all VAMP residues with side chains containing
hydrogen bond acceptors or donors were hydrogen bonded with the
LC/B. Thus, it is understood that a SNAP-25 or tagged toxin
substrate of the invention can be prepared, if desired, in which
the potential for hydrogen bonding, for example, by Ser, Thr, Tyr,
Asp, Glu, Asn or Gln residues is not diminished in the substrate as
compared to the potential for hydrogen bonding in a native protein
sensitive to cleavage by the toxin. Thus, in particular
embodiments, the present invention provides a SNAP-25 or tagged
toxin substrate in which the potential for hydrogen-bonding is not
diminished in the substrate as compared to a native protein
sensitive to cleavage by the corresponding botulinum or tetanus
toxin.
[0123] The present invention also provides a kit for determining
clostridial toxin protease activity. The kit contains a SNAP-25 or
tagged toxin substrate in a vial or other container. The kit
generally also includes instructions for use. In one embodiment, a
kit of the invention further includes as a positive control a known
amount of the botulinum or tetanus toxin, such as, without
limitation, a recombinant toxin light chain, capable of cleaving
the SNAP-25 or tagged toxin substrate included in the kit. In
another embodiment, the kit contains a SNAP-25 or tagged toxin
substrate and further includes the fluorescent or otherwise
detectable cleavage products as a positive control. A kit of the
invention may optionally include a container with buffer suitable
for clostridial toxin protease activity. As described above, the
methods of the invention can be practiced with a combination of
tagged toxin substrates. Thus, in one embodiment, the invention
provides a kit for determining clostridial toxin protease activity
that includes at least two different tagged toxin substrates.
[0124] Further provided herein are methods of determining
clostridial toxin protease activity by (a) treating with a sample,
in solution phase under conditions suitable for clostridial toxin
protease activity, a tagged toxin substrate containing (i) a
fluorescent protein; (ii) a first partner of an affinity couple;
and (iii) a clostridial toxin recognition sequence that includes a
cleavage site which intervenes between the fluorescent protein and
the first partner of the affinity couple, such that a fluorescent
cleavage product is generated when clostridial toxin is present in
the sample; (b) contacting the treated sample with a second partner
of the affinity couple, thereby forming stable complexes containing
the first and second partners of the affinity couple; and (c)
assaying the presence or amount of fluorescent cleavage product in
the treated sample, thereby determining clostridial toxin protease
activity. In one embodiment, a method of the invention is practiced
by separating the fluorescent cleavage product from the stable
complexes prior to assaying the presence or amount of fluorescent
cleavage product. A variety of fluorescent proteins can be useful
in the methods of the invention including, without limitation,
green fluorescent proteins, blue fluorescent proteins, cyan
fluorescent proteins, yellow fluorescent proteins and red
fluorescent proteins. In one embodiment, a method of the invention
is practiced using a tagged toxin substrate containing a green
fluorescent protein. First partners of an affinity couple useful in
the methods of the invention encompass, but are not limited to, a
histidine tag, glutathione-S-transferase, maltose-binding protein,
biotinylation sequence, streptavidin, S peptide, S protein, or an
epitope such as a FLAG, hemagluttinin, c-myc or AU1 epitope.
[0125] The methods of the invention can be useful for determining
clostridial toxin protease activity in any of a variety of samples.
Such samples include, but are not limited to, clarified and other
crude cell lysates; native and recombinant isolated clostridial
toxins; isolated clostridial toxin light chains; formulated
clostridial toxin products such as BOTOX.RTM. (botulinum toxin
serotype A); and foodstuffs, including raw, cooked, partially
cooked and processed foods and beverages.
[0126] In the methods of the invention, the tagged toxin substrate
is treated with a sample in solution phase. As used herein in
reference to a tagged toxin substrate, the term "in solution phase"
means that the substrate is soluble and is not constrained or
immobilized on a solid support such as a bead, column or dish.
[0127] As used herein, the term "sample" means any biological
matter that contains or potentially contains an active clostridial
toxin, or light chain or proteolytically active fragment thereof.
Thus, the term sample encompasses, but is not limited to, purified
or partially purified clostridial toxin; recombinant single chain
or dichain toxin with a naturally or non-naturally occurring
sequence; chimeric toxin containing structural elements from
multiple clostridial 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 including, without
limitation, animal, insect, bacterial and other cells engineered to
include a recombinant nucleic acid encoding a clostridial toxin or
light chain thereof; bacterial, baculoviral and yeast lysates; raw,
cooked, partially cooked or processed foods; beverages; animal
feed; soil samples; water samples; pond sediments; lotions;
cosmetics; and clinical formulations. It further is understood that
the term sample encompasses tissue samples, including, without
limitation, mammalian samples, primate samples and human samples,
and further encompassing samples such as intestinal samples, for
example, infant intestinal samples, and samples obtained from a
wound. Thus, it is understood that a method of the invention can be
useful, without limitation, to assay for clostridial toxin protease
activity in a food or beverage sample; to assay a sample from a
human or animal, for example, exposed to a clostridial toxin or
having one or more symptoms of a clostridial toxicity; to follow
activity during production and purification of clostridial toxin;
or to assay formulated clostridial toxin products, including
pharmaceuticals and cosmetics.
[0128] One skilled in the art understands that the methods of the
invention are suitable for assaying any protein or molecule with
clostridial toxin protease activity and do not rely, for example,
on the ability of the clostridial toxin to bind to a neuronal cell
or its ability to be internalized or translocated across the
membrane. Thus, the methods of the invention are suitable for
assaying for clostridial toxin protease activity of a clostridial
toxin light chain, alone, and, although useful for assaying single
or dichain heterotoxin, do not require the presence of the heavy
chain. It further is understood that the methods of the invention
are applicable to non-neuronal clostridial toxins including native
and recombinant clostridial toxins, for example, clostridial toxins
engineered to target pancreatic acinar or other non-neuronal
cells.
[0129] Depending on the clostridial toxin protease activity which
is to be assayed, a tagged toxin substrate included in a method of
the invention will incorporate one of a variety of clostridial
toxin recognition sequences. A method of the invention can be
practiced, for example, with a botulinum toxin recognition sequence
such as, without limitation, residues 134 to 206 of SEQ ID NO: 90,
or another portion of SNAP-25. A method of the invention also can
be practiced, for example, with a BoNT/A recognition sequence such
as, without limitation, a BoNT/A recognition sequence containing at
least six consecutive residues of SNAP-25, where the six
consecutive residues encompass the sequence Gln-Arg. In addition, a
method of the invention can be practiced, without limitation, with
a BoNT/B recognition sequence such as a BoNT/B recognition sequence
which includes at least six consecutive residues of VAMP, where the
six consecutive residues encompass the sequence Gln-Phe. In still
further embodiments, a method of the invention is practiced with a
tagged toxin substrate in which the recognition sequence is a
BoNT/C1 recognition sequence such as, without limitation, a BoNT/C1
recognition sequence which includes at least six consecutive
residues of syntaxin, where the six consecutive residues encompass
the sequence Lys-Ala, or a BoNT/C1 recognition sequence which
includes at least six consecutive residues of SNAP-25, where the
six consecutive residues encompass the sequence Arg-Ala.
[0130] A method of the invention also can be practiced, without
limitation, with a BoNT/D recognition sequence such as a BoNT/D
recognition sequence which includes at least six consecutive
residues of VAMP, where the six consecutive residues encompass the
sequence Lys-Leu. A method of the invention additionally can be
practiced, without limitation, with a BoNT/E recognition sequence
such as a BoNT/E recognition sequence which includes at least six
consecutive residues of SNAP-25, the six consecutive residues
encompassing the sequence Arg-Ile. In addition, a method of the
invention can be practiced, without limitation, with a BoNT/F
recognition sequence such as a BoNT/F recognition sequence which
includes at least six consecutive residues of VAMP, the six
consecutive residues encompassing the sequence Gln-Lys. A method of
the invention further can be practiced, without limitation, with a
BoNT/G recognition sequence such as a BoNT/G recognition sequence
which includes at least six consecutive residues of VAMP, where the
six consecutive residues encompass the sequence Ala-Ala. In a
further embodiment, a method of the invention is practiced with a
TeNT recognition sequence such as a TeNT recognition sequence which
includes at least six consecutive residues of VAMP, where the six
consecutive residues encompass the sequence Gln-Phe.
[0131] In the methods of the invention, a substrate can be cleaved
with any of a variety of activities. In one embodiment, a method of
the invention is practiced with a tagged toxin substrate under
conditions such that the substrate is cleaved with an activity of
at least 1 nanomole/minute/milligram toxin. In another embodiment,
a method of the invention is practiced with a tagged toxin
substrate under conditions such that the substrate is cleaved with
an activity of at least 100 nanomoles/minute/milligram toxin. In a
further embodiment, a method of the invention is practiced with a
tagged toxin substrate under conditions such that the substrate is
cleaved with an activity of at least 1000
nanomoles/minute/milligram toxin.
[0132] Any of a variety of second partners are useful in the
invention including, but not limited to, cobalt (Co.sup.2+) and
nickel (Ni.sup.2+). Furthermore, the second partner of the affinity
couple can optionally be immobilized, for example, on a column or
filter plate. In addition, a method of the invention may optionally
include the step of assaying the amount of uncleaved tagged toxin
substrate in the treated sample. It is understood that any of a
variety of samples can be assayed in a method of the invention for
determining clostridial toxin protease activity. Samples to be
assayed according to a method of the invention encompass, without
limitation, isolated clostridial toxins of any serotype; isolated
clostridial light chains; formulated clostridial toxin products
including, but not limited to, formulated BoNT/A; and whole or
partially purified cellular extracts containing one or more
recombinantly expressed clostridial toxins.
[0133] In a method of the invention, a variety of means can be used
to separate a fluorescent or otherwise detectable cleavage product
from stable complexes containing first and second partners of the
affinity couple. Separation is generally performed by specific
binding of the second partner of the affinity couple to components
within the treated sample which contain the first partner of the
affinity couple. As discussed above, by definition a fluorescent or
otherwise detectable cleavage product does not contain the first
partner of the affinity couple and, therefore, can be readily
separated from all components within a treated sample which contain
the first partner. As discussed further below, fluorescent or
otherwise detectable cleavage products are separated from stable
complexes using any of a variety of means including, but not
limited to, metal chelate affinity chromatography.
[0134] Affinity purification, including metal chelate,
immunoaffinity and other types of affinity purification techniques,
can be used to separate a fluorescent or otherwise detectable
cleavage product in a method of the invention. In one embodiment,
the first partner of the affinity couple is a histidine tag. In
another embodiment, the first partner of the affinity couple is
glutathione-S-transferase (GST). In yet another embodiment, the
first partner of the affinity couple is maltose-binding protein
(MBP). In still another embodiment, the first partner of the
affinity couple is a heterologous epitope.
[0135] In the methods of the invention, the second partner of an
affinity couple can optionally be attached to a solid support. As
used herein, the term "solid support" means an insoluble supporting
material to which a second partner can be covalently attached. The
term solid support includes, without limitation, affinity matrices
including affinity beads or gels; resins including modified
polystyrene; beads such as dextran and magnetic beads; and
carbohydrate polymers such as agarose and Sepharose.
[0136] Where the first partner of an affinity couple is a histidine
tag, metal chelate affinity chromatography (MCAC) can be useful for
separating the fluorescent or otherwise detectable cleavage
product. As used herein, the term "histidine tag" means a
consecutive series of about 6 to 10 histidine residues that
generally is solvent exposed. In one embodiment, a SNAP-25 or
tagged toxin substrate of the invention includes the 6.times.-HIS
tag HHHHHH (SEQ ID NO: 95). In another embodiment, a SNAP-25 or
tagged toxin substrate of the invention includes the 10.times.-HIS
tag HHHHHHHHHH (SEQ ID NO: 108).
[0137] Metal chelate chromatography is well known in the art as
described in Ausubel et al., supra, 10.15, Supplement 41, and
exemplified herein in Example II. Metal affinity tags useful in the
invention include, without limitation, metal affinity peptides,
which can be, for example, natural or synthetic mimics of a natural
metal-binding site. A variety of metal affinity peptides and other
metal affinity tags are known in the art as described, for example,
in Enzelberger et al., J. Chromatogr. A. 898:83-94 (2000), and
include, without limitation, 6.times.-HIS, 7.times.-HIS,
8.times.-HIS, 9.times.-HIS and 10.times.-HIS tags (Mohanty and
Weiner, Protein Expr. Purif. 33:311-325 (2004); and Grisshammer and
Tucker, Prot. Expr. Purif. 11:53-60 (1997)). In metal chelate
affinity purification, the second partner of the affinity couple is
a metal ion such as a nickel ion (Ni.sup.2+), copper ion
(Cu.sup.2+) or a cobalt ion (Co.sup.2+). As non-limiting examples,
the methods of the invention can be practiced using a bead such as
Sepharose, for example, Sepharose CL-6B; a resin; or another solid
support containing nickel or other metal ion immobilized by
iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA). Subsequent
to separation of the fluorescent or otherwise detectable cleavage
fragment (which does not contain a histidine or other metal
affinity tag) from stable complexes containing the histidine
tag-metal affinity couple, the stable complexes can optionally be
eluted from the solid support using an acidic buffer or a buffer
containing imidazole. One skilled in the art understands that a
histidine tag also can be useful for immunoaffinity separation, as
described further hereinbelow.
[0138] Glutathione-S-transferase (GST)/glutathione also can be an
affinity couple useful in the methods of the invention. In this
case, glutathione-S-transferase is incorporated into the SNAP-25 or
tagged toxin substrate as the first partner of the affinity couple.
Vectors for expression of GST-containing SNAP-25 or tagged toxin
substrates in organisms such as E. coli and Baculovirus are well
known in the art. Such vectors include the pGEX series of vectors
and are commercially available from sources such as Becton
Dickinson Biosciences and Amersham Pharmacia Biosciences
(Piscataway, N.J.). In the methods of the invention where
glutathione-S-transferase is the first partner of the affinity
couple, fluorescent or otherwise detectable cleavage fragments
(which do not contain glutathione-S-transferase) can be separated
from components within the sample that contain
glutathione-S-transferase using glutathione as the second partner
of the affinity couple. Glutathione, for example, conjugated to
agarose or other beads is commercially available from SIGMA,
Amersham Pharmacia Biosciences and other sources. Free glutathione
optionally serves to release stable complexes containing
GST-glutathione from the beads or other solid support. Affinity
chromatography using glutathione is well known in the art as
described, for example, in Smith, Methods Mol. Cell. Biol.
4:220-229 (1993), or Ausubel, supra, 2000 (see Chapter 16.7).
[0139] Maltose-binding protein/maltose also can be an affinity
couple useful in the methods of the invention. In nature,
maltose-binding protein (MBP) is encoded by the malE gene of E.
coli. Vectors are commercially available for expression of a
SNAP-25 or tagged toxin substrate containing a maltose-binding
protein as a first partner of an affinity couple. Such vectors
include pMAL vectors such as pMAL-c2e, -c2g, and -c2x, and
pMAL-p2e, -p2g and p2x and are commercially available, for example,
from sources such as New England Biolabs (Beverly, Mass.). In one
embodiment, the second partner of the affinity couple is amylose,
which is a polysaccharide consisting of maltose subunits. As a
non-limiting example, the methods of the invention can be practiced
using an amylose resin such as an agarose resin derivatized with
amylose and commercially available from New England Biolabs. Thus,
in the methods of the invention where maltose-binding protein is
the first partner of the affinity couple, fluorescent or otherwise
detectable cleavage fragments (which do not contain maltose-binding
protein) can be separated from components within the treated sample
that contain maltose-binding protein using amylose or another
maltose-containing second partner. If desired, free maltose, such
as column buffer containing 10 mM free maltose, can be used to
elute the stable complexes bound to the amylose resin. Maltose
affinity chromatography methods are routine and well known in the
art as described, for example, in Ausubel, supra, 2000 (Chapter
16.6).
[0140] Biotin-streptavidin affinity systems also can be useful in
the methods of the invention. As a non-limiting example, an 8 amino
acid streptavidin tag is known in the art (Schmidt and Skerra,
Prot. Engin. 6:109-122 (1993)) and are commercially available
(SIGMA-Genosys).
[0141] The methods of the invention also can be practiced with a
SNAP-25 or tagged toxin substrate which contains a heterologous
epitope as the first partner of the affinity couple. Such a
heterologous epitope provides a convenient means for separating the
fluorescent or otherwise detectable cleavage product. As used
herein in reference to an epitope, the term "heterologous" means an
epitope derived from a gene which is different than the gene
encoding the fused fluorescent protein or genetically encoded
detectable marker and the gene encoding the clostridial toxin
recognition sequence. Thus, for example, in a FLAG-SNAP25-GFP
tagged toxin substrate of the invention, the "FLAG" component is a
heterologous epitope which is not derived from the gene encoding
SNAP-25. A variety of heterologous epitopes are well known in the
art, including but not limited to, the FLAG epitope DYKDDDDK (SEQ
ID NO: 91; Chubet and Brizzard, BioTechniques 20:136-141 (1996);
the hemagluttinin (HA) epitope YPYDVPDYA (SEQ ID NO: 92); the c-myc
epitope EQKLISEEDL (SEQ ID NO: 93), the AU1 epitope DTYRYI (SEQ ID
NO: 94) and the 6-HIS epitope HHHHHH (SEQ ID NO: 95). One skilled
in the art understands that these and other heterologous epitopes
can be useful as first partners of an affinity couple in the
substrates and methods of the invention.
[0142] As a non-limiting example, a SNAP-25 or tagged toxin
substrate can include the FLAG tag DYKDDDDK (SEQ ID NO: 91) as the
first partner of the affinity couple. The substrate can be produced
by routine molecular methods, and the relative quantity of the
resulting detectable cleavage product determined using anti-FLAG
monoclonal antibodies commercially available, for example, from
Eastman Kodak (Rochester, N.Y.) or Berkeley Antibody Company
(BabCO; Richmond, Calif.). Similarly, the hemagluttinin (HA)
epitope YPYDVPDYA (SEQ ID NO: 92) can be engineered into a SNAP-25
or tagged toxin substrate of the invention, and the relative
quantity of the corresponding detectable cleavage fragment detected
using anti-HA antibody or antiserum obtained from BabCO (Roche
Diagnostics; Indianopolis, Ind.) or Santa Cruz Biotechnology. One
can analogously engineer into a SNAP-25 or tagged toxin substrate
the c-myc epitope EQKLISEEDL (SEQ ID NO: 93), such that the
relative quantity of corresponding detectable cleavage product can
be determined using antibody or antisera commercially available
from sources such as BabCO, Invitrogen (San Diego, Calif.), Roche
Diagnostics, SIGMA (St. Louis, Mo.) and Santa Cruz Biotechnology.
Additional heterologous epitopes useful as first partners of an
affinity couple include, without limitation, the AU1 tag DTYRYI
(SEQ ID NO: 94) recognized by a monoclonal antibody available from
BabCO, and the 6-HIS tag HHHHHH (SEQ ID NO: 95), which is
recognized by antibodies and antisera available from BabCO,
Invitrogen, SIGMA, Santa Cruz Biotechnology and other commercial
sources. One skilled in the art understands that these and other
heterologous epitopes can be conveniently used to separate a
fluorescent or otherwise detectable cleavage product in a method of
the invention.
[0143] Where the first partner of the affinity couple is a
heterologous epitope, immunoprecipitation or another immunoaffinity
separation procedure generally is used to separate the fluorescent
or otherwise detectable cleavage product in a method of the
invention. In immunoprecipitation, an antibody that recognizes the
first partner of the affinity couple is attached to a sedimentable
matrix such as, without limitation, protein A or protein G-agarose
beads or Sepharose. Low-speed centrifugation can be performed to
separate the solid-phase matrix and bound components containing the
heterologous epitope, and unbound proteins removed by washing. A
variety of immunoprecipitation protocols are routine and well known
in the art, as described, for example, in Harlow and Lane,
Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory
Press, 1988); and Ausubel, supra, 2000 (see especially Chapter 10,
Supplement 48, and Chapter 20, Supplement 46). Where the first
partner of an affinity couple is a well known heterologous epitope
such as, without limitation, a FLAG, hemagluttinin (HA), c-myc, AU1
or 6-HIS epitope, the antibodies or antiserum that specifically
bind the epitope typically are commercially available from sources
such as BabCO, Invitrogen, Roche Diagnostics, SIGMA and Santa Cruz
Biotechnology, as described hereinabove. Antibodies against these
and other heterologous epitopes also can be prepared by routine
methods as described, for example, in Harlow and Lane, supra,
1988.
[0144] An antibody useful in immunoaffinity separation of
fluorescent or otherwise detectable cleavage products can be
polyclonal or monoclonal, or a pool of monoclonal antibodies, and,
furthermore, can be an antigen-binding fragment of an antibody that
retains a specific binding activity for the first partner of the
affinity couple of at least about 1.times.10.sup.5 M.sup.-1. As
non-limiting examples, antibody fragments such as Fab, F(ab').sub.2
and F.sub.v fragments can retain specific binding activity for a
first partner of an affinity couple and, thus, can be useful in the
invention. Furthermore, immunoaffinity separation can be performed
with a non-naturally occurring antibody or fragment containing, at
a minimum, one V.sub.H and one V.sub.L domain, for example, a
chimeric antibody, humanized antibody or single chain Fv fragment
(scFv) that specifically binds the first partner of the affinity
couple. Such a non-naturally occurring antibody can be constructed
using solid phase peptide synthesis, produced recombinantly, or
obtained, for example, by screening combinatorial libraries
consisting of variable heavy chains and variable light chains as
described by Borrebaeck (Ed.), Antibody Engineering (Second
edition) New York: Oxford University Press (1995)). If desired, an
antibody can be attached to a solid support for immunoaffinity
separation. Such solid supports include, without limitation,
Sepharose, which is an insoluble, large-pore size chromatographic
matrix. In one embodiment, an antibody is attached to Sepharose
CL-4B, a 4% cross-linked agarose. Elution can be performed, for
example, using brief exposure to high or low pH (Ausubel, supra,
Chapter 10.11A, 2000).
[0145] As discussed further below, a variety of conditions suitable
for clostridial toxin protease activity are useful in the methods
of the invention. For example, conditions suitable for clostridial
toxin protease activity can be provided such that at least 10% of
the substrate is cleaved. Similarly, conditions suitable for
clostridial toxin protease activity can be provided such that at
least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the SNAP-25
or tagged toxin substrate is cleaved, or such that 100% of the
SNAP-25 or tagged toxin substrate is cleaved. In one embodiment,
the conditions suitable for clostridial toxin protease activity are
selected such that the assay is linear. In another embodiment,
conditions suitable for clostridial toxin protease activity are
provided such that at least 90% of the SNAP-25 or tagged toxin
substrate is cleaved. In a further embodiment, conditions suitable
for clostridial toxin protease activity are provided such that at
most 25% of the SNAP-25 or tagged toxin substrate is cleaved. In
yet further embodiments, conditions suitable for clostridial toxin
protease activity are provided such that at most 5%, 10%, 15% or
20% of the SNAP-25 or tagged toxin substrate is cleaved.
[0146] In the methods of the invention, a sample is treated with a
SNAP-25 or tagged toxin substrate in solution phase under
conditions suitable for clostridial toxin protease activity.
Exemplary conditions suitable for clostridial 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 clostridial toxin
protease activity can depend, in part, on the specific clostridial
toxin type or subtype being assayed and the purity of the toxin
preparation. Conditions suitable for clostridial 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 clostridial toxin protease activity also
can include, if desired, dithiothreitol, .beta.-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, an isolated
clostridial toxin 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 SNAP-25 or tagged toxin substrate.
[0147] Clostridial toxins are zinc metalloproteases, and a source
of zinc, such as zinc chloride or zinc acetate, typically in the
range of 1 to 500 .mu.M, for example, 5 to 10 .mu.M can be
included, if desired, as part of the conditions suitable for
clostridial toxin protease activity. One skilled in the art
understands that zinc chelators such as EDTA generally are excluded
from a buffer for assaying clostridial toxin protease activity.
[0148] Conditions suitable for clostridial toxin protease activity
can optionally include a detergent such as TWEEN-20, which can be
used, for example, in place of bovine serum albumin. TWEEN-20 can
be provided, for example, in the range of 0.001% to 10% (v/v)
Tween-20, or in the range of 0.01% to 1.0% (v/v) Tween-20. In one
embodiment, TWEEN-20 is provided at a concentration of 0.1% (v/v;
see Example II).
[0149] Conditions suitable for clostridial toxin protease activity
can optionally include a detergent such as TWEEN-20.RTM.
(polyoxyethylene (20) sorbitan monolaureate), which can be used,
for example, in place of bovine serum albumin. TWEEN-20
(polyoxyethylene (20) sorbitan monolaureate) can be provided, for
example, in the range of 0.001% to 10% (v/v) TWEEN-20.RTM.
(polyoxyethylene (20) sorbitan monolaureate), or in the range of
0.01% to 1.0% (v/v) Tween-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate). In one embodiment, TWEEN-20 (polyoxyethylene (20)
sorbitan monolaureate) is provided at a concentration of 0.1% (v/v;
see Example II).
[0150] The amount of SNAP-25 or tagged toxin substrate can be
varied in a method of the invention. A SNAP-25 or tagged toxin
substrate can be supplied, for example, at a concentration of 1
.mu.M to 500 .mu.M, 1 .mu.M to 50 .mu.M, 1 .mu.M to 30 .mu.M, 5
.mu.M to 20 .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 SNAP-25 or tagged toxin substrate or the
amount of sample can be limited, if desired, such that the assay is
linear. In one embodiment, a method of the invention relies on a
SNAP-25 or tagged toxin substrate concentration of less than 100
.mu.M. In further embodiments, a method of the invention relies on
a SNAP-25 or tagged toxin substrate concentration of less than 50
.mu.M or less than 25 .mu.M. In a further embodiment, a method of
the invention relies on a SNAP-25 or tagged toxin substrate
concentration of 10 .mu.M to 20 .mu.M. If desired, a linear assay
also can be performed by mixing SNAP-25 or tagged toxin substrate
with corresponding, "unlabeled" substrate which lacks the green
fluorescent protein, or fluorescent protein or genetically encoded
detectable marker. The appropriate dilution can be determined, for
example, by preparing serial dilutions of SNAP-25 or tagged toxin
substrate in the corresponding unlabeled substrate.
[0151] The concentration of purified or partially purified
clostridial toxin assayed in a method of the invention generally is
in the range of about 0.1 .mu.M to 100 nM, for example, 1 .mu.M to
2000 .mu.M, 1 .mu.M to 200 .mu.M, 1 .mu.M to 50 .mu.M, 1 to 200 nM,
1 to 100 nM or 3 to 100 nM toxin, which can be, for example,
purified native or recombinant light chain or dichain toxin or
formulated clostridial toxin product containing human serum albumin
and excipients. In particular embodiments, the concentration of
purified or partially purified recombinant BoNT/A or BoNT/E light
chain or dichain or formulated toxin product is in the range of 1
pM to 2000 pM, 10 pM to 2000 pM, 20 pM to 2000 pM, 40 pM to 2000
pM, or 1 pM to 200 pM. In further embodiments, the concentration of
purified or partially purified recombinant BoNT/C light chain or
dichain or formulated toxin product is in the range of 1 to 200 nM,
4 to 100 nM, 10 to 100 nM or 4 to 60 nM. One skilled in the art
understands that the concentration of purified or partially
purified clostridial toxin will depend on the serotype of the toxin
assayed, as well as the purity of the toxin, the presence of
inhibitory components, and the assay conditions. It is additionally
understood that purified, partially purified or crude samples can
be diluted to within a convenient range for assaying for
clostridial toxin protease activity against a standard curve.
Similarly, it is understood that a sample can be diluted, if
desired, such that the assay for toxin protease activity is
linear.
[0152] Conditions suitable for clostridial 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.
[0153] Assay times can be varied as appropriate by the skilled
artisan and generally depend, in part, on the concentration, purity
and activity of the clostridial toxin. Assay times generally vary,
without limitation, in the range of about 15 minutes to about 5
hours. As non-limiting examples, exemplary assay times include
incubation, for example, at 37.degree. C. for 30 minutes, 45
minutes, 60 minutes, 75 minutes or 90 minutes (see Example III). In
particular embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 95% or 100% of the SNAP-25 or tagged toxin substrate is
cleaved. In further embodiments, the protease reaction is stopped
before more than 5%, 10%, 15%, 20%, 25% or 50% of the SNAP-25 or
tagged toxin substrate is cleaved. Protease reactions can be
terminated by the appropriate reagent, which generally depends on
the fluorescent protein or other detectable marker in the
substrate. As a non-limiting example, a protease reaction based on
a GFP-containing substrate can be terminated by addition of a
terminating reagent such as guanidinium chloride, for example, to a
final concentration of 1 to 2 M as in Example II. Protease
reactions also can be terminated by addition of H.sub.2SO.sub.4;
addition of about 0.5 to 1.0 M 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, if desired, prior to
contacting the treated sample with a second partner of the affinity
couple.
[0154] As a non-limiting example, conditions suitable for
clostridial toxin protease activity such as BoNT/A protease
activity can be incubation at 37.degree. C. for 90 minutes in a
buffer containing 50 mM HEPES (pH 7.2), 10 .mu.M ZnCl.sub.2, 10 mM
DTT, and 0.1% (v/v) TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate) with 10-16 .mu.M substrate (see Example II). If
desired, BoNT/A, particularly dichain BoNT/A, can be preincubated
with dithiothreitol, for example, for 20 or 30 minutes before
addition of substrate. As a further non-limiting example,
conditions suitable for BoNT/A protease activity can be incubation
at 37.degree. C. in a buffer such as 30 mM HEPES (pH 7.3)
containing a reducing agent such as 5 mM dithiothreitol; and a
source of zinc such as 25 .mu.M zinc chloride (approximately 7 nM;
Schmidt and Bostian, supra, 1997). BSA in the range of 0.1 mg/ml to
10 mg/ml, for example, 1 mg/ml BSA, also can be included when a
sample is treated with a SNAP-25 or tagged toxin substrate (Schmidt
and Bostian, supra, 1997). As still a further non-limiting example,
conditions suitable for clostridial toxin protease activity, for
example BoNT/B activity, can be incubation in 50 mM HEPES, pH 7.4,
with 10 .mu.M zinc chloride, 1% fetal bovine serum and 10 mM
dithiothreitol, with incubation for 90 minutes at 37.degree. C.
(Shone and Roberts, Eur. J. Biochem. 225:263-270 (1994); Hallis et
al., supra, 1996); or can be, for example, incubation in 40 mM
sodium phosphate, pH 7.4, with 10 mM dithiothreitol, optionally
including 0.2% (v/v) Triton X-100, with incubation for 2 hours at
37.degree. C. (Shone et al., supra, 1993). Conditions suitable for
tetanus toxin protease activity or other clostridial toxin protease
activity can be, for example, incubation in 20 mM HEPES, pH 7.2,
and 100 mM NaCl for 2 hours at 37.degree. C. with 25 .mu.M peptide
substrate (Cornille et al., supra, 1994).
[0155] In a method of the invention for determining clostridial
toxin protease activity, a sample is treated with a tagged toxin
substrate containing (i) a fluorescent protein or other genetically
encoded detectable marker; (ii) a first partner of an affinity
couple; and (iii) a clostridial toxin recognition sequence that
includes a cleavage site which intervenes between the fluorescent
protein and the first partner of the affinity couple. If desired, a
second tagged toxin substrate can be included; this second
substrate contains a second fluorescent protein or other
genetically encoded detectable marker and a second first partner of
an affinity couple as well as a second clostridial toxin
recognition sequence including a second cleavage site that is
cleaved by a different clostridial toxin than the toxin that
cleaves the first cleavage site within the first clostridial toxin
recognition sequence. The fluorescent protein or other genetically
encoded detectable marker and the first partner of an affinity
couple in the second substrate can be the same or different from
those in the first substrate. In this way, a single sample can be
conveniently assayed for the presence of multiple clostridial
toxins.
[0156] It is understood that one can assay for any combination of
clostridial toxins, for example, two, three, four, five, six,
seven, eight, nine, ten or more clostridial toxins. One can assay,
for example, any combination of two, three, four, five, six, seven
or eight of TeNT, BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F
and BoNT/G. For example, seven substrates, each containing the same
fluorescent or other genetically encoded detectable protein and
same first partner of an affinity couple flanking a BoNT/A, BoNT/B,
BoNT/C1, BoNT/D, BoNT/E, BoNT/F or BoNT/G recognition sequence can
be treated with a sample in solution phase under conditions
suitable for botulinum toxin protease activity before contacting
the treated sample with a second partner of the affinity couple.
The presence of fluorescent cleavage product is indicative of
clostridial toxin protease activity of at least one botulinum
toxin. Such an assay can be useful, for example, for assaying food
samples or tissue samples for the presence of any botulinum toxin
and can be combined, if desired, with one or more subsequent assays
for individual botulinum toxins or specific combinations of
botulinum toxins.
[0157] In another embodiment, a single sample is assayed for two or
more different clostridial toxins using two or more different
tagged toxin substrates with each substrate containing a different
fluorescent protein or other genetically encoded detectable marker.
The use of multiple substrates can be useful for extending the
dynamic range of the assay, as described, for example, in U.S. Pat.
No. 6,180,340. As an example of the use of multiple tagged toxin
substrates, a single sample can be assayed for BoNT/A and BoNT/B
protease activity using a first tagged toxin substrate containing a
green fluorescent protein and a BoNT/A recognition sequence, and a
second tagged toxin substrate containing a red fluorescent protein
and a BoNT/B recognition sequence. If desired, the two substrates
can utilize the same first partner of an affinity couple, such as a
histidine tag. Subsequent to contacting the treated sample with a
second partner of the affinity couple and separating fluorescent
cleavage product from stable complexes, a green fluorescent
cleavage product is indicative of BoNT/A protease activity while a
red fluorescent cleavage product is indicative of BoNT/B protease
activity, and both green and red fluorescent cleavage products are
indicative of BoNT/A and BoNT/B cleavage products.
[0158] Multiple substrates also can be used in the methods of the
invention to extend the range of the assay. For example, at least
two tagged toxin substrates are used together at different
dilutions; the substrates have different fluorescent proteins or
other genetically encoded detectable markers and, therefore, are
separately detectable, but have recognition sequences for the same
clostridial toxin. In one embodiment, otherwise identical tagged
toxin substrates with different fluorescent proteins or other
genetically encoded detectable markers are used together at
different dilutions to extend the dynamic range of a method of the
invention.
[0159] One or more controls may optionally be utilized in the
methods of the invention. A control substrate typically is the same
SNAP-25 or tagged toxin substrate which is treated with a defined
sample containing one or more clostridial toxins; the same SNAP-25
or tagged toxin substrate which is not treated with any sample; or
a non-cleavable form of the SNAP-25 or tagged toxin substrate. One
skilled in the art understands that a variety of control substrates
are useful in the methods of the invention and that a control
substrate can be a positive control substrate or a negative control
substrate. A control substrate can be, for example, a negative
control such as a similar or identical substrate that is contacted
with a similar sample that does not contain active clostridial
toxin, or that is not contacted with any sample. A control cleavage
product, similar or identical to the fluorescent or otherwise
detectable cleavage product, also can be useful in the methods of
the invention.
[0160] It is understood that the methods of the invention can be
automated and, furthermore, can be configured in a high-throughput
or ultra high-throughput format using, for example, 96-well,
384-well or 1536-well plates. As one example, fluorescence emission
can be detected using Molecular Devices FLIPR instrumentation
system (Molecular Devices; Sunnyvale, Calif.), which is designed
for 96-well plate assays (Schroeder et al., J. Biomol. Screening
1:75-80 (1996)). FLIPR utilizes a water-cooled 488 nm argon ion
laser (5 watt) or a xenon arc lamp and a semiconfocal optimal
system with a charge-coupled device (CCD) camera to illuminate and
image the entire plate. The FPM-2 96-well plate reader (Folley
Consulting and Research; Round Lake, Ill.) also can be useful in
detecting fluorescence emission in the methods of the invention.
One skilled in the art understands that these and other automated
systems with the appropriate spectroscopic compatibility such as
the ECLIPSE cuvette reader (Varian-Cary; Walnut Creek, Calif.), the
SPECTRA.sub.max GEMINI XS (Molecular Devices) and other systems
from, for example, Perkin Elmer can be useful in the methods of the
invention.
[0161] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Expression and Characterization of Recombinant GFP-Snap-25
Substrates
[0162] This example describes construction of plasmids for
expression of GFP-SNAP25.sub.(134-206) and SNAP25.sub.(134-206)-GFP
substrates as well as control substrates containing modified
cleavage sites.
[0163] Two GFP substrates containing the same components, but
present in the opposite orientations were designed and expressed.
Each substrate was a fusion protein consisting of green fluorescent
protein (GFP), murine SNAP-25 residues 134-206, and a polyhistidine
affinity tag (6.times.His), with each component separated by
peptide linkers. As described further below, the substrates were
designed such that the GFP and polyhistidine tag were fused to
opposite termini of SNAP25.sub.(134-206). The fusion protein
substrates were designated GFP-SNAP25 and SNAP25-GFP.
A. Construction of pQE50/BirASNAP.sub.(128-206)
[0164] The SNAP-25 sequence was obtained from pT25FL, a plasmid
which contains the full-length mouse SNAP-25 gene inserted in frame
with the 3' terminus of the glutathione-S-transferase (GST) gene
(GST-SNAP25.sub.(1-26)), provided by Professor Dolly (O'Sullivan et
al., J. Biol. Chem. 274:36897-36904 (1999)). The SNAP-25 sequence
from pT25FL was incorporated into a second expression vector, which
was designed to have a BirAsp signal sequence for biotinylation and
a polyhistidine affinity tag fused to the N-terminus of residues
134 to 206 of SNAP-25 (BirAsp-polyHis-SNAP25.sub.(134-206), denoted
"BA-SNAP"). The DNA sequence encoding SNAP25.sub.(134-206) was
generated by PCR amplification of the appropriate region of the
pT25FL plasmid with PCR primers 5'-GCT AGA TCT CGA GTT MC CAC TTC
CCA GCA TCT TTG-3' (SEQ ID NO: 104; antisense) and 5'-ATC CGG AGG
GTA ACA AAC GAT GCC-3' (SEQ ID NO: 101; sense) to produce a
SNAP25.sub.(134-126) PCR product containing a Bgl II restriction
site (PCR product A).
[0165] The BirAsp sequence, a natural substrate for biotinylation,
as well as a polyhistidine affinity tag, were engineered for fusion
upstream and in frame with the SNAP25.sub.(134-206) sequence using
synthetic oligonucleotides SEQ ID NOS: 102 and 103, which contained
a 20 bp complementary region. These oligonucleotides, 5'-CGA ATT
CCG CGG GCC ACC ATG GGA GGA GGA CTG AAC GAC ATC TTC GAG GCT CM MG
ATC-3' (SEQ ID NO: 102; sense; Sac II site underlined) and 5'-TCG
TTT GTT ACC CTC CGG ATA TGA TGA TGA TGA TGA TGA TGA TGG GAT CCA TGC
CAC TCG ATC TTT TGA GCC TCG MG A-3' (SEQ ID NO: 103; antisense),
were annealed, and the single strand overhangs filled by PCR
amplification to yield PCR product B.
[0166] The two double stranded PCR products containing the coding
sequences for SNAP25.sub.(134-206), denoted PCR product A, and
BirAsp and polyhistidine, denoted PCR product B, were denatured and
annealed. The 20 bp complementary sequence in the two gene
fragments is shown in italics in PCR primers SEQ ID NO: 101 and SEQ
ID NO: 103). After filling in the overhangs by PCR, the product was
amplified with primers SEQ ID NO: 102 and SEQ ID NO: 104. The
resulting PCR product, which encoded
BirAsp-polyHis-SNAP25.sub.(134-206) (designated "BA-SNAP"), was
digested with SacII and BglII, and the isolated gene insert ligated
into pQBI25fA2 vector digested with SacII and BamHI, to yield
plasmid pNTP12 (pQBI25fA2 containing BA-SNAP).
[0167] For expression and purification from E. coli, the BA-SNAP
gene was transferred into a pTrc99A plasmid (Amersham Pharmacia
Biotech). The BA-SNAP gene was isolated from pNTP12 by digestion
with NcoI and XhoI followed by gel purification. Separately, the
pTrc99A plasmid was digested with NcoI and SalI, and the isolated
vector ligated to the BA-SNAP gene to yield plasmid pNTP14 (pTrc99A
containing BA-SNAP).
[0168] For cloning of the BA-SNAP gene into plasmid pQE-50, the
A-SNAP fragment was PCR amplified from pNTP14 with primer SEQ ID
NO: 104 and primer SEQ ID NO: 105 (5'-CGA AGA TCT GGA GGA CTG AAC
GAC ATC TTC-3' (sense; Bgl II site underlined)). After digestion
with Bgl II and XhoI, the amplified PCR product was ligated into
vector pQE-50, which had been digested with BamH I and Sal I. The
resulting plasmid, which represents pQE50 containing BA-SNAP, was
designated pNTP26.
B. Construction of GFP-SNAP-25 Expression Vectors
[0169] Plasmids encoding the green fluorescent protein (GFP) fusion
protein substrates were prepared by modifying vector pQBI T7-GFP
(Quantum Biotechnologies; Carlsbad, Calif.) as described below. The
plasmid maps are shown in FIGS. 8A and 9A below. The nucleic acid
and predicted amino acid sequence for the GFP-SNAP25.sub.(134-206)
and SNAP25.sub.(134-206)-GFP substrates are shown in FIGS. 8B and
9B, respectively.
[0170] Plasmid pQBI GFP-SNAP25.sub.(134-206) was constructed in two
phases as follows. First, vector pQBI T7-GFP was PCR-modified to
remove the stop codon at the 3' terminus of the GFP-coding sequence
and to insert the coding sequence for a portion of the peptide
linker separating GFP from the SNAP-25 fragment. Second, a DNA
fragment coding for SNAP-25.sub.(134-206) was PCR amplified from
pNTP26 using PCR primers designed to incorporate the coding
sequence for the remainder of the peptide linker fused 5' to the
SNAP-25.sub.(134-206) gene and a 6.times.His affinity tag fused 3'
of the gene. The resultant PCR product was cloned into the modified
pQBI vector described above to yield the pQBI
GFP-SNAP25.sub.(134-206) plasmid (see FIG. 8A) for expression of
GFP-SNAP25.sub.(134-206)-6.times.His.
[0171] Plasmid pQBI SNAP25.sub.(134-206)-GFP was constructed as
follows. Plasmid pQBI SNAP25.sub.(134-206)-GFP, shown in FIG. 9B,
was constructed by subcloning a PCR amplified gene containing the
BirAsp biotinylation sequence, a poly-His affinity tag, and SNAP25
residues 134-206 into pQBI T7-GFP. The entire BirAsp, 6.times.His,
and SNAP25.sub.(134-206) gene from pNTP26 was PCR amplified using
primers designed to incorporate the coding sequence for a fusion
protein linker 3' of the amplified gene and to facilitate fusion to
the 5' terminus of the GFP gene, yielding a single gene for
expression of BirAsp-6.times.His-SNAP25.sub.(134-206)-linker-GFP as
shown in FIG. 9B.
C. Construction of Vectors Encoding SNAP-25/GFP Expression Vector
Variants
[0172] Modification of vector pQBI GFP-SNAP25.sub.(134-206) to
create the Arg198Ala and Arg180Asp analogues was performed using
primers 5'-GATGAAGCCAA CCAAGCTGCAACAAAGATGCTG-3' (SEQ ID NO: 106;
"SNAP25(R198A)") and 5'-CGCCAGATCGACGATATCATGGAGAAGGCTG-3' (SEQ ID
NO: 107; "SNAP25(R180D)") along with their complementary
sequences.
[0173] For each pair of primers, six 50 .mu.L PCR reactions were
assembled containing 5 .mu.L 10.times.Pfu Buffer (Stratagene; La
Jolla, Calif.), 1 .mu.L dNTPs (12.5 mM each; Promega; Madison,
Wis.), 1 .mu.L Pfu Turbo DNA polymerase (Stratagene; hot start
addition), varying concentrations of template DNA (10 to 100 ng
pQBI GFP-SNAP25.sub.134-206) and each primer at a final
concentration of 0.2 .mu.M. The reactions were brought to a final
volume of 50 .mu.L with nuclease-free water. Following incubation
at 95.degree. C. for 2 minutes, 25 cycles of amplification were
performed (95.degree. C. for 1 minute; 60.degree. C. for 30
seconds; and 72.degree. C. for 12 minutes), followed by a final
72.degree. C. extension for 7 minutes.
[0174] Following thermocycling, 1 .mu.L DpnI restriction enzyme
(Stratagene) was added to each reaction and incubated for one hour
at 37.degree. C. to digest template DNA. The reactions were
purified by QIAquick kit (Qiagen; Valencia, Calif.) and analyzed by
agarose gel electrophoresis. All but one of the reactions produced
full-length plasmid. Sequencing of the candidate plasmids revealed
one Arg180Asp variant and two Arg198Ala variants containing the
desired changes.
D. Expression and Purification of GFP-SNAP25 Substrate
[0175] The expression vectors described above were transformed into
E. coli BL21 (DE3) cells (Novagen; Madison, Wis.; or Invitrogen;
Carlsbad, Calif.) or into E. coli BL21-CodonPlus.RTM. (DE3)-RIL
cells (Stratagene) containing the T7 RNA polymerase gene.
Transformed cells were selected on LB(amp) plates overnight at
37.degree. C. Single colonies were used to inoculate 1-3 mL starter
cultures which were in turn used to inoculate 0.5 to 1.0 L
cultures. The large cultures were grown at 37.degree. C. with
shaking until A.sub.595 reached 0.5-0.6, at which time they were
removed from the incubator and were allowed to cool briefly. After
induction of protein expression with 1 mM IPTG, GFP-SNAP25
substrate was expressed from the pQBI GFP-SNAP25.sub.(134-206)
plasmid overnight with shaking at 16.degree. C. in order to
facilitate folding of the GFP moiety. Cells from 250 mL aliquots of
the expression cultures were collected by centrifugation (30
minutes, 6,000.times.g, 4.degree. C.) and stored at -80.degree. C.
until needed.
[0176] Substrates were purified at 4.degree. C. by a two-step
procedure involving IMAC purification, followed by a de-salting
step to remove imidazole, typically yielding greater than 150 mg/L
of purified substrate, as follows. Cell pellets from 250 mL
cultures were each resuspended in 7-12 mL Column Binding Buffer (25
mM HEPES, pH 8.0; 500 mM NaCl; 1 mM .beta.-mercaptoethanol; 10 mM
imidazole), lysed by sonication (1 minute 40 seconds in 10-second
pulses at 38% amplitude), and clarified by centrifugation (16000
rpm, 4.degree. C., 1 hour). Affinity resin (3-5 mL Talon SuperFlow
Co.sup.2+ per cell pellet) was equilibrated in a glass or
disposable column support (Bio-Rad) by rinsing with 4 column
volumes of sterile ddH.sub.2O and 4 column volumes of Column
Binding Buffer. Clarified lysate was applied to the column in one
of two ways: (1) Lysate was added to the resin and batch bound by
horizontal incubation for 1 hour with gentle rocking or (2) Lysate
was applied to the vertical column and allowed to enter the column
slowly by gravity flow. Following batch binding only, the column
was righted and the solution drained, collected, and passed over
the resin again. In both cases, after the lysate had been applied,
the column was washed with 4-5 column volumes of Column Binding
Buffer. In some cases, the column was further washed with 1-2
column volumes of Column Wash Buffer (25 mM HEPES, pH8.0; 500 mM
NaCl; 1 mM .beta.-mercaptoethanol; 20 mM imidazole). Protein was
eluted with 1.5 to 2.0 column volumes of Column Elution Buffer (25
mM HEPES, pH 8.0; 500 mM NaCl; 1 mM .beta.-mercaptoethanol; 250 mM
imidazole), which was collected in fractions of .about.1.4 mL. The
green fractions were combined and desalted by FPLC (BioRad Biologic
DuoLogic, QuadTec UV-Vis detector) with a HiPrep 26/10 size
exclusion column (Pharmacia) and an isocratic mobile phase of
chilled Fusion Protein Desalting Buffer (50 mM HEPES, pH 7.4,
4.degree. C.) at a flow rate of 10 mL/minute. Desalted protein was
collected as a single fraction, concentrated in an Apollo 20-mL
concentrator (QMWL 10 kDa; Orbital Biosciences), and the
concentration determined using a BioRad Protein Assay. The
GFP-SNAP25 substrate was monomeric as demonstrated by
size-exclusion chromatography. The protein solution was
subsequently divided into 500 .mu.L aliquots, flash-frozen with
liquid nitrogen and stored at -80.degree. C. Once defrosted, a
working aliquot was stored at 4.degree. C., protected from
light.
E. Characterization of GFP-SNAP25 Substrates
[0177] As shown in FIGS. 10A-C, the specificity of Type A toxin for
the Q197-R198 scissile bond of GFP-SNAP25 was verified by SDS-PAGE
and Western blot analysis of substrate cleaved by rLC/A. Similarly
it was demonstrated that proteolysis of GFP-SNAP25 with BoNT/E
yields the expected GFP-SNAP25(134-180) product (FIGS. 10D-F).
These results demonstrate that a synthetic substrate containing GFP
and a histidine tag as well as a portion of SNAP-25 containing a
clostridial toxin recognition sequence and cleavage site can be an
effective substrate for the relevant clostridial toxin.
[0178] SDS-PAGE and Western blot analysis of BoNT/A and BoNT/E
proteolytic reactions were performed as follows.
GFP-SNAP25.sub.134-206 substrate (0.4 mg/mL) was combined with
either single-chain native BoNT/E (50.0 .mu.g/mL), or rLC/A (0.1
.mu.g/mL) in toxin reaction buffer (50 mM HEPES pH 7.4, 0.1
.mu.g/mL BSA, 10 .mu.M ZnCl.sub.2, 10 mM DTT). The reactions were
incubated at 37.degree. C., with aliquots removed after incubation
for 0, 5, 10, 15, 30, and 60 minutes and quenched by addition to
gel loading buffer. Reaction mixtures were analyzed by SDS-PAGE
(10% Bis-Tris MOPS) and staining with Sypro Ruby (Bio-Rad) or were
transferred to nitrocellulose membranes and probed with antibodies
specific for GFP, SNAP25.sub.134-197, or SNAP25.sub.134-180.
F. Expression and Identification of Cell Lysates Containing Active
Recombinant BoNT Types A and E
[0179] Crude, clarified BL21-Codon Plus cell lysates were prepared
as follows. Chemically competent E. coliBL21-CodonPlus.RTM.
(DE3)-RIL cells (Stratagene) were transformed with a pET vector to
provide kanamycin resistance, spread on LB/kan (50 .mu.g/mL) plates
and incubated overnight at 37.degree. C. A 3 mL culture grown from
a single colony was used to inoculate a 175 mL culture, which was
incubated overnight with shaking at 37.degree. C. The cells were
pelleted by centrifugation, resuspended in 10 mL 25 mM HEPES
buffer, pH 7.4, and lysed by sonication (1 minute 40 seconds in
10-sec pulses at 38% amplitude). The lysate was cleared by
centrifugation (16,000 rpm, 4.degree. C., 1 hour), divided into 0.4
mL aliquots, flash-frozen with liquid nitrogen and stored at
-80.degree. C. The protein content of the cleared lysate was
determined to be 0.7 mg/mL by the BioRad Protein Assay.
[0180] TOP10.RTM. cell lysates were prepared as follows. Joanne
Wang provided a 20 mL culture of ampicillin-resistant E. coli TOP10
cells (Invitrogen). Cells were pelleted by centrifugation and
resuspended in 2 mL Toxin Reaction Buffer without DTT (50 mM HEPES,
pH 7.4; 0.1% (v/v) TWEEN-20.RTM. (polyoxyethylene (20) sorbitan
monolaureate); 10 .mu.M ZnCl.sub.2). Cells were lysed by sonication
(1 minute 40 seconds in 10-sec pulses at 38% amplitude). Two 200
.mu.L aliquots were withdrawn before clarifying the lysate by
centrifugation (14000 rpm, 4.degree. C., 30 minutes). All but the
working aliquots of the crude and clarified lysates were
flash-frozen with liquid nitrogen and stored at -80.degree. C.
[0181] Single colonies of BL21 (DE3) cells transformed with
plasmids potentially encoding the gene for active BoNT/A or BoNT/E
were used to inoculate 1.0 mL cultures in OVERNIGHT EXPRESS.RTM.
autoinduction medium (Novagen); the BoNT/A cultures contained 100
.mu.g/mL ampicillin, and the BoNT/E cultures contained 50 .mu.g/mL
kanamycin. Safe-Lock tubes equipped with membrane lids (2 mL;
Eppendorf; Westbury, N.Y.) were used. Cultures were grown at
37.degree. C., 1400 rpm, in a Thermomixer R (Eppendorf) until
cloudy (about 3 hours for Type A and 7 hours for Type E), at which
point the temperature was reduced, and the cultures incubated at
16.degree. C. overnight. Cells were collected by centrifugation (15
minutes at 6,000.times.g, 4.degree. C.) and stored at -80.degree.
C. until needed.
[0182] To identify cultures expressing active toxin, clarified cell
lysates were prepared and tested in the GFP-SNAP25 assay. Cell
pellets were defrosted on ice and each was lysed (20 minutes at
23.degree. C., 300 rpm in the Thermomixer R) with 500 .mu.L
BUGBUSTER.RTM. Protein Extraction Reagent containing 25 U/mL
benzonase nuclease and, for the Type A cell pellets, 1.2 KU/mL
RLYSOZYME.RTM. and 1.times. Protease Inhibitor Cocktail III (all
four reagents from Novagen). Lysates were clarified by
centrifugation (20 minutes at 16000 rpm, 4.degree. C.) and the
supernatant solutions transferred to fresh microcentrifuge tubes.
The assay reactions contained 10 .mu.L clarified lysate and 10
.mu.M (Type A) or 15 .mu.M (Type E) GFP-SNAP25 substrate in a total
volume of 50 .mu.L Toxin Reaction Buffer (50 mM HEPES, pH 7.2; 10
.mu.M ZnCl.sub.2; 10 mM DTT; 0.1% (v/v) TWEEN-20.RTM.
(polyoxyethylene (20) sorbitan monolaureate)). Two control
reactions were assembled, each containing dH.sub.2O in place of the
lysate and one containing rLC/E at a final concentration of 0.01
.mu.g/mL. All reactions were assembled in triplicate and incubated
at 37.degree. C., for 40 minutes (Type A reactions) or 1 hour (Type
E reactions). Reactions were quenched and processed as described
below under general procedures for the GFP-SNAP25 assay.
EXAMPLE II
GFP-SNAP25 Fluorescence Release Assay
[0183] This example describes specific proteolysis of
GFP-SNAP25.sub.(134-206) and quantification of proteolysis using a
fluorescence release assay.
A. Overview of GFP-SNAP25 Fluorescence Release Assay
[0184] A summary of the GFP-SNAP25 fluorescence release assay is
illustrated in FIG. 10G. Processing of the reaction mixture was
dependent on the presence of a polyhistidine affinity tag which
facilitates immobilized metal affinity chromatography (IMAC)
separation of unreacted GFP-SNAP25 from endopeptidase generated
GFP-SNAP25.sub.(134-197) cleavage product. As an overview, the
treated substrate was processed as follows. First, a solution phase
reaction was initiated by the addition of substrate to the
appropriate light chain (zinc metalloprotease) or pre-reduced
botulinum neurotoxin (serotype A, C, or E). Following incubation
for the desired period of time under conditions described further
below, the reaction was quenched by the addition of guanidinium
chloride to a final concentration of 1-2 M. After termination of
the reaction, unreacted substrate was separated from the
endopeptidase-generated GFP-containing product
(GFP-SNAP25.sub.(134-197)) on IMAC resin. The reaction mixture was
applied to a spin column or a 96-well filter plate containing
Co.sup.2+ resin in order to bind the polyhistidine-tagged species.
The GFP-SNAP25.sub.(134-197) fragment that is freed from the
polyhistidine affinity tag by proteolysis passes through the resin
in the first "flow-through" fraction, while the unreacted substrate
remains bound to the resin. Following collection of the
flow-through fraction, the resin was washed with buffer to remove
residual GFP-SNAP25.sub.(134-197) before unreacted GFP-SNAP25
substrate was eluted with imidazole. Collection of both the
GFP-SNAP25.sub.(134-197) cleavage product as well as the GFP-SNAP25
substrate facilitates quantification of both product and unreacted
substrate by fluorescence. The relative fluorescence of
endopeptidase product (and, if desired, unreacted substrate) can
then be plotted against the toxin concentration.
B. Solution Phase Clostridial Proteolysis Reactions
[0185] Clostridial toxin protease reactions were performed as
follows. Toxin, recombinant toxin, or recombinant LC/A or LC/E was
diluted to twice the desired reaction concentration with 2.times.
Toxin Reaction Buffer (100 mM HEPES, pH 7.2 or 7.4; 20 .mu.M
ZnCl.sub.2; 20 mM DTT; 0.2% (v/v) BSA or TWEEN-20.RTM.
(polyoxyethylene (20) sorbitan monolaureate)) and added to black
v-bottom 96-well plates (Whatman; Clifton, N.J.) or microcentrifuge
tubes in aliquots equal to one-half the final reaction volume. Each
light chain or toxin sample was pre-incubated at 37.degree. C. for
20 minutes. Prior to initiation of the reactions, GFP-SNAP25
substrate was diluted with sterile ddH.sub.2O to twice the desired
reaction concentration and warmed to 37.degree. C. Reactions were
initiated by addition of substrate to each well or tube in aliquots
equal to one-half the final reaction volume. The final
concentration of substrate in standard reactions was 10-16 .mu.M.
Reaction vessels were sealed, protected from light, and incubated
at 37.degree. C. The incubation period was generally 90 minutes,
except that time course reactions were frequently run for longer
periods; reactions were run in triplicate. At the end of the
incubation period or at specified time points, reactions or
reaction aliquots were quenched by the addition of 8 M guanidine
hydrochloride to a final concentration of 1-2 M and subsequently
processed as described below.
C. Separation of Fluorescent Cleavage Product
[0186] Samples were processed as described below with all steps
either performed manually with a UniVac.RTM. vacuum manifold
(Whatman) for elution at -15 in Hg or, with the exception of the
addition of resin to the filter plate, were performed on a
Biomek-FX liquid handling system (Beckman/Coulter; Fullerton,
Calif.). The required number of wells in a 96-well filter plate
(400 .mu.L or 800 .mu.L wells, 0.45 .mu.m filter, long drip;
Innovative Microplate; Chicopee, Mass.) were loaded with 75 .mu.L
of Talon.TM. Superflow Co.sup.2+ affinity resin (Becton Dickinson
Biosciences). Resin storage buffer was removed by vacuum, and the
resin conditioned by rinsing with 4 column volumes ddH.sub.2O and 4
column volumes Assay Rinse Buffer (50 mM HEPES, pH 7.4). The last
aliquot of Assay Rinse Buffer was eluted from the resin immediately
prior to use.
[0187] Following quenching with guanidine hydrochloride, reaction
solutions were transferred to filter plate wells containing
conditioned Co.sup.2+ resin, where they were incubated at room
temperature for 15 minutes. The reaction solutions were then
eluted, collected in a black, flat-bottom 96-well plate (BD
Falcon), passed over the resin beds twice more and collected after
the final pass. Each resin bed was then rinsed with 2.times.135
.mu.L Assay Rinse Buffer that was eluted into the plate containing
the eluant reaction solution, which contains the GFP product.
[0188] In some cases, unreacted substrate was collected after
washing the resin beds with 3.times.250 .mu.L Assay Rinse Buffer.
Unreacted substrate was then eluted from the resin beds with 260
.mu.L Assay Elution Buffer 500 (50 mM HEPES, pH 7.4; 500 mM
imidazole) and collected in a black, flat-bottom 96-well plate (BD
Falcon).
[0189] The fluorescence of the reaction flow-through and imidazole
eluant solutions was quantified with a SpectraMax Gemini XS
spectrophotometer (Molecular Devices; .lamda..sub.EX 474 nm;
.lamda..sub.Em 509 nm; 495 nm cutoff filter).
D. Results of GFP-SNAP25 Fluorescence Release Assays
[0190] Results of the GFP-SNAP25 fluorescence release assays are
summarized below. The ability to easily measure unreacted starting
material provided an internal control for the reactions and served
to demonstrate that substrate was completely converted to product
(see FIG. 11C as an example). Proteolytic activity of recombinant
light chain type A (rLC/A) and botulinum neurotoxin serotypes A and
E was detected at picomolar concentrations. The enzymatic activity
of BoNT/C complex was also detected at low nanomolar
concentrations, consistent with literature reports for serotype C
requiring the presence of membranes for activity and having poor in
vitro activity in general (Vaidyanathan et al., J. Neurochem
72:327-337 (1999); Foran et al., Biochemistry 35:2630-2636 (1996);
Blasi et al., EMBO J. 12:4821-4828 (1993)).
[0191] Recombinant type A light chain (rLC/A) activity. The
activity of rLC/A was efficiently measured with the GFP-SNAP25
fluorescence release assay as shown in FIG. 11A. For reactions in
which the substrate concentration was 16 .mu.M, rLC/A at a
concentration range of 9 to 1,250 pM yielded reaction products with
relative fluorescence units (RFUs) from 730 to over 24,000. As
further demonstrated in FIG. 11A, a significant signal was measured
at a rLC/A concentration of 36 pM.
[0192] 150 kDa BoNT/A (pure A) toxin activity. The activity of
native BoNT/A toxin was also efficiently measured with the
GFP-SNAP25 fluorescence release assay (see FIG. 11B). For reactions
in which the substrate concentration was 16 .mu.M, native pure
BoNT/A at a concentration range of 17 to 1,000 pM yielded reaction
products with relative fluorescence units from 4,600 to almost
24,000. FIG. 11B further shows that a significant signal was
measured at a pure BoNT/A concentration of 17 pM.
[0193] 900 kDa BoNT/A (bulk) toxin activity. The BoNT/A complex was
very efficient at cleaving GFP-SNAP25 substrate, with 6 .mu.M of
bulk BoNT/A toxin complex yielding a signal that was 19 fold above
background (see FIG. 11C). For reactions in which the substrate
concentration was 16 .mu.M, BoNT/A at a concentration range of 3 to
89 .mu.M yielded reaction products with (relative fluorescence
units) from 2,600 to over 60,000 (FIG. 11C).
[0194] 146 kDa BoNT/E (pure E) toxin activity. Unlike BoNT/A,
single chain (SC) BoNT/E is not nicked by Clostridia to form the
activated dichain form. Activation of native single chain BoNT/E
toxin can be accomplished by exogenous treatment of toxin with
trypsin. As shown in FIGS. 12A and 12B, respectively, native single
chain and dichain BoNT/E both cleaved the GFP-SNAP25 substrate.
Trypsin nicking of single chain BoNT/E to yield the dichain form
substantially increased the proteolytic activity of serotype E.
[0195] As shown in FIG. 12A, for reactions in which the substrate
concentration was 16 .mu.M, native single-chain BoNT/E at a
concentration range of 2 to 21 nM yielded reaction products with
relative fluorescence units from approximately 3,800 to 22,000. As
shown in FIG. 12B, the specific activity of native dichain BoNT/E
under the same reaction conditions was much greater. At a
concentration range of 17 to 1,546 pM, dichain BoNT/E yielded
reaction products with relative fluorescence units from
approximately 1,200 to 19,000.
[0196] BoNT/C complex activity. As discussed above, BoNT/C is known
to cleave both syntaxin and SNAP-25 in vivo, with the BoNT/C
cleavage site within the GFP-SNAP substrate residing at
Arg198-Ala199 of SNAP25. GFP-SNAP substrate was assayed as a
substrate for type C by incubation with BoNT/C complex. As shown in
FIG. 12C, GFP-SNAP can detect BoNT/C activity; however, it is not
detected as readily as BoNT/A or /E activity. For reactions in
which the substrate concentration was 16 .mu.M, native BoNT/C at a
concentration range of 4 to 60 nM yielded reaction products with
relative fluorescence units from approximately 3,800 to 22,000,
consistent with literature reports of poor in vitro activity for
serotype C and reports that BoNT/C may require the presence of
membranes for efficient enzymatic activity (Vaidyanathan et al.,
supra, 1999; Foran et al., supra, 1996; Blasi et al., supra,
1993).
EXAMPLE III
Variation of Assay Conditions with Recombinant GFP-SNAP-25
Substrate
[0197] This example describes variation and optimization of the
GFP-SNAP25 fluorescence release assay.
A. Assay Optimization: BSA vs. Tween-20
[0198] Initially, GFP-SNAP assays were conducted in Toxin Reaction
Buffer containing bovine serum albumin (BSA) as a protein
carrier/stabilizer (50 mM Hepes, pH 7.4, 10 .mu.M ZnCl.sub.2, 10 mM
DTT, and 0.1 mg/mL BSA). The reaction buffers for some botulinum
neurotoxin assays contain the detergent TWEEN-20' (polyoxyethylene
(20) sorbitan monolaureate), rather than BSA. An investigation
comparing the effect of these protein stabilizers on BoNT reactions
revealed that serotypes A, C, and E all have significantly higher
activity in the presence of 0.1% (v/v) TWEEN-20.RTM.
(polyoxyethylene (20) sorbitan monolaureate) as compared to BSA.
These results indicate that the use of TWEEN-20.degree.
(polyoxyethylene (20) sorbitan monolaureate) in place of BSA
results in higher activity for BoNT/A, /C and /E .
B. Assay Optimization: pH
[0199] The pH of the protease reaction buffers was varied within
the range of 7.0-8.2. Bulk A toxin was most active at pH 7.2 while
type E dichain was most active at pH 7.0. pH values below 7 were
not assayed since fluorescence of the GFP fusion protein substrate
is quenched under acidic conditions (Ekong et al., Dev. Animal Vet.
Sci. 27:1039-1044 (1997)). Although the activity of bulk A toxin is
known to be dependent on the release of toxin and light chain from
the complex, a process which is most efficient at elevated pH
(Hallis et al., J. Clin. Microbiol. 34:1934-1938 (1996)), the
results obtained with the GFP-SNAP25 substrate agree with those
indicating that the optimum pH for rLC/A expressed from a synthetic
gene is pH 7.2 and that activity drops fairly quickly on either
side of this optimum. In contrast, the pH preference of type E
toxin was not as pronounced as that of Bulk A; type E activity was
not completely eliminated even at pH 8.2.
[0200] pH profiles of bulk A toxin and pure E dichain toxin were
determined as follows. The general procedures for the GFP-SNAP25
assay described above were followed to determine the optimal toxin
reaction pH, except as noted. Seven 2.times. Toxin Reaction Buffer
solutions were prepared at pH 7.00, 7.20, 7.41, 7.60, 7.80, 8.01,
and 8.24. These buffers were used to prepare the toxin dilutions.
The final reaction concentrations of the toxins were 89 pM native
BoNT/A complex and 203 pM native pure E dichain. Reactions were
quenched after a 90 minute incubation.
C. Assay Optimization: Dithiothreitol Dependence
[0201] Using bulk A toxin, it was observed that the lack of a
pre-incubation period with 10 mM dithiothreitol simply resulted in
a delay in the production of cleavage product. In contrast, the
absence of dithiothreitol resulted in essentially complete loss of
activity.
[0202] GFP-SNAP25 time-course assays of bulk BoNT/A toxin for
dithiothreitol (DTT) dependence were performed as follows. The
general procedures for the GFP-SNAP25 assay described above were
followed, except as noted below, for testing the dependence of bulk
A activity upon DTT and the requirement for a pre-incubation
period. An initial dilution of bulk A toxin was made in 2.times.
Toxin Reaction Buffer without DTT. Aliquots were removed from this
solution to prepare additional dilutions, one into Toxin Reaction
Buffer containing DTT and the other into the same buffer without
DTT. Buffer solutions were pre-warmed to 30.degree. C. Four types
of reactions plus one substrate-only control, each in triplicate,
were assembled. Two sets of reactions contained DTT and two did
not, with just one set of each type pre-incubated for 20 minutes at
30.degree. C. prior to initiation of the reactions. The time
elapsed from transfer of the bulk A from the stock solution to the
initiation of the "no pre-incubation" reactions was 1.5 minutes.
The substrate-only control reactions did not contain DTT but were
pre-incubated. The final reaction volume was 400 .mu.L and
contained 44 pM bulk A and 16 .mu.M GFP-SNAP25 substrate. Aliquots
of 50 .mu.L were removed from each reaction and quenched with 20
.mu.L guanidine HCl at 15, 30, 45, 60, and 90 minute time
points.
D. Specificity of Toxin Cleavage Demonstrated Using Mutant
GFP-SNAP25 Substrate with Altered Scissile Bonds
[0203] As discussed above, the fusion protein substrate used in the
GFP-SNAP25 assay contains residues 134-206 of SNAP-25. BoNT/A
cleaves SNAP-25 between residues Ala 197 and Arg 198, while BoNT/C
cleaves the neighboring peptide bond between residues Arg 198 and
Ala 199. The conversion of Arg 198 to alanine has been shown to
eliminate detectable hydrolysis by BoNT/A in certain assays
(Schmidt et al., FEBS Left 435:61-64 (1998)) and likely also
eliminates proteolysis by BoNT/C. The Arg 198 to Ala change was
therefore introduced into the GFP-SNAP25 substrate by site-directed
mutagenesis to create a control fusion protein denoted
GFP-SNAP25(R198A).
[0204] The cleavage site for BoNT/E resides between residues Arg
180 and Ile 181; however, no single-residue mutation has been shown
to eliminate hydrolysis. An Arg 180 to aspartate mutation was
selected for a possible control substrate, due to the charge
reversal as well as steric differences introduced by the change.
The GFP-SNAP substrate analogue containing this mutation is denoted
GFP-SNAP(R180D).
[0205] Fusion protein substrate mutants R198A#2, R198A#4 and R180D
were expressed and purified essentially as described above for the
standard GFP-SNAP25.sub.(134-206) substrate. Mutant substrates were
then tested under GFP-SNAP25 assay conditions with relatively high
concentrations of toxins (4.5 nM rLC/A, 6.8 nM native pure E
dichain, and 60 nM BoNT/C complex). As demonstrated in FIG. 13,
despite high toxin concentrations, there was virtually no cleavage
of the R198A mutants by rLC/A; furthermore, cleavage by the BoNT/C
complex was reduced to approximately 14% of the level seen with the
GFP-SNAP25 substrate, which translates to .about.4.times. the
background signal. Proteolysis by BoNT/E of the R180D mutant was
significantly reduced to approximately 15% of the standard
substrate proteolysis, although the signal remained greater than
11.times. the background signal in spite of the steric differences
and reversal of charge introduced into the substrate at the site of
the scissile bond.
[0206] Analysis of R180D proteolysis by type E toxin was repeated
to confirm that the result was reproducible and to verify cleavage
at the Asp180/Ile181 bond. SDS-PAGE analysis of the reaction
products confirmed that the R180D mutant was partially cleaved by
Type E, and that the larger fragment produced has the same apparent
molecular weight as that produced by proteolysis of the standard
GFP-SNAP25 substrate.
[0207] In addition to the toxin proteolysis experiments, trypsin
digestion of the substrates was performed. The GFP portion of the
substrates was expected to remain intact as GFP has been shown to
be resistant to proteases other than pronase (U.S. Pat. No.
5,965,699). As expected, essentially all of the GFP was released by
trypsin digestion.
[0208] Trypsin digestion of GFP-SNAP25 substrates was performed as
follows. Spin columns and filters (35 .mu.m pore size; MoBiTec;
Goettingen, Germany) were packed with 5 .mu.L of agarose bead-bound
trypsin (24.1 U/mL of packed gel; Sigma). Fusion protein substrates
(GFP-SNAP25 and mutants R198A#4 and R180D) were diluted to a
concentration of 30 .mu.g/15 .mu.L in Digest Buffer (20 mM HEPES,
pH 7.4; 300 mM NaCl). Reactions were run in duplicate and initiated
by addition of 15 .mu.L of the substrate solution to a spin column
containing the trypsin gel. After incubation for one hour at
30.degree. C., reaction products were eluted by centrifugation and
collected in 1.7 mL microcentrifuge tubes. Each resin bed was
rinsed with 40 .mu.L Digest Buffer, which was eluted and collected
in the same tube as the reaction solution. Samples were
subsequently processed according to the filter-plate processing
methods described above.
E. Cleavage of Fusion Protein Substrates by Endogenous Proteases in
Crude Lysates
[0209] Substrate mutants R180D and R198A serve as substrate
controls to distinguish toxin activity in a cell lysate from the
proteolytic activity of endogenous proteases. Essentially, any
non-toxin proteolysis in a lysate would be reflected in the signal
from control substrates, while the GFP-SNAP25 signal would reflect
the combined activity of toxin and endogenous proteases.
[0210] Lysates from two types of E. coli cells were assayed. The
first was a lysate from BL21-CODONPLUS.RTM.(DE3)-RIL, a protease
deficient cell line transformed to express rLC/A and the fusion
protein substrates. Reactions were assembled to mimic the
GFP-SNAP25 assay conditions, except that either 5 .mu.L or 20 .mu.L
of clarified lysate was included in lieu of toxin. As shown in FIG.
14A, there was insignificant proteolysis of any of the substrates.
Only the signals for the reactions containing R180D exceed the
background signals typically seen in the GFP-SNAP25 assay. Cell
lysates from TOP10.RTM. cells, which are not protease deficient,
were also assayed. In these experiments, 20 .mu.L of clarified
lysate was included, and once again the level of hydrolysis was
negligible (FIG. 14B). These results indicate that the proteolysis
of GFP-SNAP25 substrates is specific to clostridial toxins and is
not due to other proteases endogenous to cell lystates.
[0211] Proteolysis of fusion protein substrates in crude, clarified
cell lysates was performed as follows. Reactions containing 5 .mu.L
or 20 .mu.L of crude, clarified lysate from E. coli
BL21-CODONPLUA.RTM. (DE3)-RIL cells (Stratagene) or E. coli
TOP10.RTM. cells (Invitrogen) were assembled and initiated by
addition of GFP substrate (GFP-SNAP25, R198A, or R180D) to a final
concentration of 16 .mu.M. Substrate dilutions were prepared with
2.times. Toxin Reaction Buffer (100 mM HEPES, pH 7.4; 20 .mu.M
ZnCl.sub.2; 20 mM DTT; 0.2% (v/v) TWEEN-20.RTM. (polyoxyethylene
(20) sorbitan monolaureate)), and an additional 5 .mu.L of the
2.times. Toxin Reaction Buffer was included in each reaction so
that the final concentration would be approximately 1.times. Toxin
Reaction Buffer. In all cases final reaction volumes were 50 .mu.L,
and reactions were run in triplicate. For control reactions,
sterile ddH.sub.2O was included in place of cell lysate. Reactions
were incubated for one hour at 37.degree. C., quenched with 15
.mu.L 8M guanidine hydrochloride, and processed according to the
filter-plate processing method described above.
F. Kinetic Data for rLC/A
[0212] Several reaction and processing conditions were explored in
order to determine whether the GFP-SNAP25 fluorescence release
assay was sensitive over a substrate concentration range flanking
the KM, and whether the required substrate concentrations can be
accommodated under the standard processing conditions. Data were
obtained by running a series of time course assays at a single
toxin concentration over a range of substrate concentrations. The
reaction conditions and processing details such as the length of
the reactions and the resin volumes were varied among assays. For
these initial tests, all the reactions contained 178 pM rLC/A and
the substrate concentration was varied from 2.5-80 .mu.M.
[0213] The process of kinetic analysis included non-linear fitting
of curves to plots of the data, determination of initial substrate
velocities from these curves, and lastly plotting the initial
velocities against substrate concentration to estimate of v.sub.max
and K.sub.m. The initial plot provides a preliminary K.sub.m value
of 4.6 .mu.M at this toxin concentration (FIGS. 15 and 16).
Alternatively, the assay can be run at many times this substrate
concentration.
[0214] Published Type A toxin kinetic constants, most of which were
determined with HPLC-based assays, are shown in Table 6.
[0215] Time-course assays of rLC/A for kinetic analyses were
performed using a final toxin concentration of 0.01 .mu.g/mL (0.18
nM), while the substrate concentration was varied from 2.5-80
.mu.M. Three reactions plus one to three substrate-only controls
were assembled at each substrate concentration as described under
general procedures for the GFP-SNAP25 Assay, and the reactions were
incubated for two to seven hours. Aliquots of 50 .mu.L (30 .mu.L
for the 80 .mu.M substrate reactions) were withdrawn over the
course of the reactions, beginning at the 4 minute point, and
quenched with 20 .mu.L 8M guanidine hydrochloride. The quenched
samples were processed as described above under general procedures
for the GFP-SNAP25 assay.
TABLE-US-00006 TABLE 6 Published Kinetic Constants Enzyme Substrate
K.sub.m (.mu.M) k.sub.cat (s-1) Reference BoNT/A
rSNAP25.sub.(1-206) 79 .+-. 8 0.009 Schmidt et al., J Prot. Chem
16: 19-26 (1997) BoNT/A rSNAP25.sub.(137-206) 353 .+-. 17 0.02
Schmidt et al., supra, 1997 BoNT/A Various 17-mer peptides 580-5000
1.8-56 Schmidt et al., FEBS Lett. 435: 61-64, (1998) BoNT/A 17-mer
peptide 5000 .+-. 500 4.7 .+-. 0.5 U.S. Pat. No. 5,965,699 rLC/A
17-mer peptide 1100 .+-. 100 23 .+-. 1 U.S. Pat. No. 5,965,699
rLC/A FIA 96 .+-. 10 7.2 .+-. 0.4 U.S. Pat. No. 5,965,699
G. GFP-SNAP25 Fluorescence Release Assay of Single Vials of
Botox.RTM.
[0216] From the results described above, the GFP-SNAP assay is
sensitive enough to assay the contents of single vials of
BOTOX.RTM. (botulinum toxin serotype A). However, BOTOX.RTM.
(botulinum toxin serotype A) did not significantly cleave
GFP-SNAP25 substrate under the standard conditions described above.
Notably, a salt content (.about.0.88 mg NaCl/vial) as well as a
large amount of HSA (.about.0.5 mg/vial) are present in BOTOX.RTM.
(botulinum toxin serotype A). A dialysis step to remove the salt
resulted in increased background fluorescence and did not produce
significant proteolysis. To remove the HSA, which might interfere
with proteolysis or binding of His-tagged species to the
purification resin, the BOTOX.RTM. (botulinum toxin serotype A)
vial contents were dialyzed using a Teflon-coated dialysis unit
with 100,000 MWCO membrane. The contents of a vial were resuspended
in 100 .mu.L dH.sub.2O, and, following dialysis, approximately 1/5
of the toxin from a vial was included in each of the final
reactions, assuming that 100% of the toxin was transferred from the
vial and recovered following dialysis. The contents of two vials
were assayed in a total of six reactions, and a placebo control
vial was also assayed in triplicate. As shown in FIG. 17, the
BOTOX.RTM. (botulinum toxin serotype A) signal was readily
detectable at 15-fold above background. These results indicate that
the high NaCl and HSA content of formulated BOTOX.RTM. (botulinum
toxin serotype A) product can interfere with the GFP-SNAP25
fluorescence release assay. These results further indicate that,
following removal of NaCl and human serum albumin through dialysis
or another method, BOTOX.RTM. (botulinum toxin serotype A) or other
formulated toxin product can be assayed using the GFP-SNAP25
fluorescence release assay disclosed herein.
[0217] GFP-SNAP25 Assays of BOTOX.RTM. (botulinum toxin serotype A)
were performed as follows. The contents of two vials of BOTOX.RTM.
(botulinum toxin serotype A).RTM. were dissolved, each in 100 .mu.L
sterile dH.sub.2O, and transferred to the same dialysis unit (Fast
Spin Dializer, Harvard Apparatus, 100,000 MWCO). The contents of a
single placebo vial were also dissolved in 100 .mu.L sterile
dH.sub.2O and transferred to a dialysis unit of the same type but
smaller volume. The toxin solution was dialyzed against 2.times.1 L
BOTOX.RTM. (botulinum toxin serotype A) Dialysis Buffer (50 mM
HEPES, pH 7.2; 10 .mu.M ZnCl.sub.2), and the placebo solution was
dialyzed against 2.times.500 mL BOTOX.RTM. (botulinum toxin
serotype A).RTM. Dialysis Buffer, with a total dialysis time of one
hour at room temperature. Following dialysis, 140 .mu.L of the
BOTOX.RTM. (botulinum toxin serotype A) solution was combined with
35 .mu.L BOTOX.RTM. (botulinum toxin serotype A).RTM. Reaction
Buffer (50 mM HEPES, pH 7.2; 10 .mu.M ZnCl.sub.2; 0.17% (v/v)
TWEEN-20.RTM. (polyoxyethylene (20) sorbitan monolaureate); 16 mM
DTT) pre-warmed to 30.degree. C.; 80 .mu.L of the placebo solution
was combined with 20 .mu.L of the BOTOX.RTM. (botulinum toxin
serotype A) Reaction Buffer. Both solutions were preincubated at
30.degree. C. for 20 minutes. The GFP-SNAP25 dilution (to 40 .mu.M)
was prepared with the BOTOX.RTM. (botulinum toxin serotype A)
Reaction Buffer. Reactions were initiated by combining 25 .mu.L of
either the BOTOX.RTM. (botulinum toxin serotype A) or placebo
solution with 25 .mu.L of the substrate solution. Six BOTOX.RTM.
(botulinum toxin serotype A) reactions and three placebo control
reactions were initiated and incubated at 30.degree. C. for 3
hours, 5 minutes. Reactions were quenched with 20 .mu.L 8 M
guanidine hydrochloride and processed as described in the above
general procedures for the GFP-SNAP25 assay.
[0218] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0219] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
11318PRTArtificial Sequencesynthetic construct 1Glu Ala Asn Gln Arg
Ala Thr Lys 1 52206PRTHomo sapiens 2Met Ala Glu Asp Ala Asp Met Arg
Asn Glu Leu Glu Glu Met Gln Arg 1 5 10 15Arg Ala Asp Gln Leu Ala
Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu
Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu
Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile
Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu
Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys 85 90
95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val
100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln
Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp
Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser
Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met
Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile
Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu Ala
Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200
20538PRTArtificial Sequencesynthetic construct 3Gly Ala Ser Gln Phe
Glu Thr Ser 1 54116PRTHomo sapiens 4Met Ser Ala Thr Ala Ala Thr Ala
Pro Pro Ala Ala Pro Ala Gly Glu 1 5 10 15Gly Gly Pro Pro Ala Pro
Pro Pro Asn Leu Thr Ser Asn Arg Arg Leu 20 25 30Gln Gln Thr Gln Ala
Gln Val Asp Glu Val Val Asp Ile Met Arg Val 35 40 45Asn Val Asp Lys
Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu Asp 50 55 60Asp Arg Ala
Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser65 70 75 80Ala
Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met Met 85 90
95Ile Ile Leu Gly Val Ile Cys Ala Ile Ile Leu Ile Ile Ile Ile Val
100 105 110Tyr Phe Ser Ser 11558PRTArtificial Sequencesynthetic
construct 5Asp Thr Lys Lys Ala Val Lys Trp 1 568PRTArtificial
Sequencesynthetic construct 6Arg Asp Gln Lys Leu Ser Glu Leu 1
57116PRTRattus sp. 7Met Ser Ala Thr Ala Ala Thr Val Pro Pro Ala Ala
Pro Ala Gly Glu 1 5 10 15Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu
Thr Ser Asn Arg Arg Leu 20 25 30Gln Gln Thr Gln Ala Gln Val Asp Glu
Val Val Asp Ile Met Arg Val 35 40 45Asn Val Asp Lys Val Leu Glu Arg
Asp Gln Lys Leu Ser Glu Leu Asp 50 55 60Asp Arg Ala Asp Ala Leu Gln
Ala Gly Ala Ser Gln Phe Glu Thr Ser65 70 75 80Ala Ala Lys Leu Lys
Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met Met 85 90 95Ile Ile Leu Gly
Val Ile Cys Ala Ile Ile Leu Ile Ile Ile Ile Val 100 105 110Tyr Phe
Ser Thr 11588PRTArtificial Sequencesynthetic construct 8Gln Ile Asp
Arg Ile Met Glu Lys 1 598PRTArtificial Sequencesynthetic construct
9Glu Arg Asp Gln Lys Leu Ser Glu 1 5108PRTArtificial
Sequencesynthetic construct 10Glu Thr Ser Ala Ala Lys Leu Lys 1
5118PRTArtificial Sequencesynthetic construct 11Gly Ala Ser Gln Phe
Glu Thr Ser 1 512206PRTMus musculus 12Met Ala Glu Asp Ala Asp Met
Arg Asn Glu Leu Glu Glu Met Gln Arg 1 5 10 15Arg Ala Asp Gln Leu
Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val
Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp
Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln
Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75
80Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys Asn Lys Leu Lys
85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly
Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu
Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn
Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val
Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp
Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg
Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile 180 185 190Asp Glu
Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly 195 200
20513212PRTDrosophila sp. 13Met Pro Ala Asp Pro Ser Glu Glu Val Ala
Pro Gln Val Pro Lys Thr 1 5 10 15Glu Leu Glu Glu Leu Gln Ile Asn
Ala Gln Gly Val Ala Asp Glu Ser 20 25 30Leu Glu Ser Thr Arg Arg Met
Leu Ala Leu Cys Glu Glu Ser Lys Glu 35 40 45Ala Gly Ile Arg Thr Leu
Val Ala Leu Asp Asp Gln Gly Glu Gln Leu 50 55 60Asp Arg Ile Glu Glu
Gly Met Asp Gln Ile Asn Ala Asp Met Arg Glu65 70 75 80Ala Glu Lys
Asn Leu Ser Gly Met Glu Lys Cys Cys Gly Ile Cys Val 85 90 95Leu Pro
Cys Asn Lys Ser Gln Ser Phe Lys Glu Asp Asp Gly Thr Trp 100 105
110Lys Gly Asn Asp Asp Gly Lys Val Val Asn Asn Gln Pro Gln Arg Val
115 120 125Met Asp Asp Arg Asn Gly Met Met Ala Gln Ala Gly Tyr Ile
Gly Arg 130 135 140Ile Thr Asn Asp Ala Arg Glu Asp Glu Met Glu Glu
Asn Met Gly Gln145 150 155 160Val Asn Thr Met Ile Gly Asn Leu Arg
Asn Met Ala Leu Asp Met Gly 165 170 175Ser Glu Leu Glu Asn Gln Asn
Arg Gln Ile Asp Arg Ile Asn Arg Lys 180 185 190Gly Glu Ser Asn Glu
Ala Arg Ile Ala Val Ala Asn Gln Arg Ala His 195 200 205Gln Leu Leu
Lys 21014203PRTCarassius auratus 14Met Ala Asp Glu Ala Asp Met Arg
Asn Glu Leu Thr Asp Met Gln Ala 1 5 10 15Arg Ala Asp Gln Leu Gly
Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu
Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu
Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly Met 50 55 60Asp Gln Ile
Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu Thr Asp65 70 75 80Leu
Gly Asn Leu Cys Gly Leu Cys Pro Cys Pro Cys Asn Lys Leu Lys 85 90
95Gly Gly Gly Gln Ser Trp Gly Asn Asn Gln Asp Gly Val Val Ser Ser
100 105 110Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala Ile
Ser Gly 115 120 125Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu
Asn Glu Met Asp 130 135 140Glu Asn Leu Glu Gln Val Gly Ser Ile Ile
Gly Asn Leu Arg His Met145 150 155 160Ala Leu Asp Met Gly Asn Glu
Ile Asp Thr Gln Asn Arg Gln Ile Asp 165 170 175Arg Ile Met Asp Met
Ala Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala 180 185 190Asn Gln Arg
Ala Thr Lys Met Leu Gly Ser Gly 195 20015212PRTStrongylocentrotus
purpuratus 15Met Glu Asp Gln Asn Asp Met Asn Met Arg Ser Glu Leu
Glu Glu Ile 1 5 10 15Gln Met Gln Ser Asn Met Gln Thr Asp Glu Ser
Leu Glu Ser Thr Arg 20 25 30Arg Met Leu Gln Met Ala Glu Glu Ser Gln
Asp Met Gly Ile Lys Thr 35 40 45Leu Val Met Leu Asp Glu Gln Gly Glu
Gln Leu Asp Arg Ile Glu Glu 50 55 60Gly Met Asp Gln Ile Asn Thr Asp
Met Arg Glu Ala Glu Lys Asn Leu65 70 75 80Thr Gly Leu Glu Lys Cys
Cys Gly Ile Cys Val Cys Pro Trp Lys Lys 85 90 95Leu Gly Asn Phe Glu
Lys Gly Asp Asp Tyr Lys Lys Thr Trp Lys Gly 100 105 110Asn Asp Asp
Gly Lys Val Asn Ser His Gln Pro Met Arg Met Glu Asp 115 120 125Asp
Arg Asp Gly Cys Gly Gly Asn Ala Ser Met Ile Thr Arg Ile Thr 130 135
140Asn Asp Ala Arg Glu Asp Glu Met Asp Glu Asn Leu Thr Gln Val
Ser145 150 155 160Ser Ile Val Gly Asn Leu Arg His Met Ala Ile Asp
Met Gln Ser Glu 165 170 175Ile Gly Ala Gln Asn Ser Gln Val Gly Arg
Ile Thr Ser Lys Ala Glu 180 185 190Ser Asn Glu Gly Arg Ile Asn Ser
Ala Asp Lys Arg Ala Lys Asn Ile 195 200 205Leu Arg Asn Lys
21016249PRTGallus gallus 16Met Ala Glu Asp Ala Asp Met Arg Asn Glu
Leu Glu Glu Met Gln Arg 1 5 10 15Arg Ala Asp Gln Leu Ala Asp Glu
Ser Leu Glu Ser Thr Arg Arg Met 20 25 30Leu Gln Leu Val Glu Glu Ser
Lys Asp Ala Gly Ile Arg Thr Leu Val 35 40 45Met Leu Asp Glu Gln Gly
Glu Gln Leu Asp Arg Val Glu Glu Gly Met 50 55 60Asn His Ile Asn Gln
Asp Met Lys Glu Ala Glu Lys Asn Leu Lys Asp65 70 75 80Leu Gly Lys
Cys Cys Gly Leu Phe Ile Cys Pro Cys Asn Lys Leu Lys 85 90 95Ser Ser
Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn Gln Asp Gly Val 100 105
110Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg Glu Gln Met Ala
115 120 125Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp Ala Arg
Glu Asn 130 135 140Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile
Ile Gly Asn Leu145 150 155 160Arg His Met Ala Leu Asp Met Gly Asn
Glu Ile Asp Thr Gln Asn Arg 165 170 175Gln Ile Asp Arg Ile Met Glu
Lys Leu Ile Pro Ile Lys Pro Gly Leu 180 185 190Met Lys Pro Thr Ser
Val Gln Gln Arg Cys Ser Ala Val Val Lys Cys 195 200 205Ser Lys Val
His Phe Leu Leu Met Leu Ser Gln Arg Ala Val Pro Ser 210 215 220Cys
Phe Tyr His Gly Ile Tyr Leu Leu Gly Leu His Thr Cys Thr Tyr225 230
235 240Gln Pro His Cys Lys Cys Cys Pro Val 245 17116PRTMus musculus
17Met Ser Ala Thr Ala Ala Thr Val Pro Pro Ala Ala Pro Ala Gly Glu 1
5 10 15Gly Gly Pro Pro Ala Pro Pro Pro Asn Leu Thr Ser Asn Arg Arg
Leu 20 25 30Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met
Arg Val 35 40 45Asn Val Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser
Glu Leu Asp 50 55 60Asp Arg Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln
Phe Glu Thr Ser65 70 75 80Ala Ala Lys Leu Lys Arg Lys Tyr Trp Trp
Lys Asn Leu Lys Met Met 85 90 95Ile Ile Leu Gly Val Ile Cys Ala Ile
Ile Leu Ile Ile Ile Ile Val 100 105 110Tyr Phe Ser Thr
11518116PRTBos taurus 18Met Ser Ala Thr Ala Ala Thr Ala Pro Pro Ala
Ala Pro Ala Gly Glu 1 5 10 15Gly Gly Pro Pro Ala Pro Pro Pro Asn
Leu Thr Ser Asn Arg Arg Leu 20 25 30Gln Gln Thr Gln Ala Gln Val Asp
Glu Val Val Asp Ile Met Arg Val 35 40 45Asn Val Asp Lys Val Leu Glu
Arg Asp Gln Lys Leu Ser Glu Leu Asp 50 55 60Asp Arg Ala Asp Ala Leu
Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser65 70 75 80Ala Ala Lys Leu
Lys Arg Lys Tyr Trp Trp Lys Asn Leu Lys Met Met 85 90 95Ile Ile Leu
Gly Val Ile Cys Ala Ile Ile Leu Ile Ile Ile Ile Val 100 105 110Tyr
Phe Ser Ser 11519114PRTXenopus laevis 19Met Ser Ala Pro Ala Ala Gly
Pro Pro Ala Ala Ala Pro Gly Asp Gly 1 5 10 15Ala Pro Gln Gly Pro
Pro Asn Leu Thr Ser Asn Arg Arg Leu Gln Gln 20 25 30Thr Gln Ala Gln
Val Asp Glu Val Val Asp Ile Met Arg Val Asn Val 35 40 45Asp Lys Val
Leu Glu Arg Asp Thr Lys Leu Ser Glu Leu Asp Asp Arg 50 55 60Ala Asp
Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Thr Ser Ala Ala65 70 75
80Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Met Lys Met Met Ile Ile
85 90 95Met Gly Val Ile Cys Ala Ile Ile Leu Ile Ile Ile Ile Val Tyr
Phe 100 105 110Ser Thr 20104PRTStrongylocentrotus purpuratus 20Met
Ala Ala Pro Pro Pro Pro Gln Pro Ala Pro Ser Asn Lys Arg Leu 1 5 10
15Gln Gln Thr Gln Ala Gln Val Asp Glu Val Val Asp Ile Met Arg Val
20 25 30Asn Val Asp Lys Val Leu Glu Arg Asp Gln Ala Leu Ser Val Leu
Asp 35 40 45Asp Arg Ala Asp Ala Leu Gln Gln Gly Ala Ser Gln Phe Glu
Thr Asn 50 55 60Ala Gly Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Cys
Lys Met Met65 70 75 80Ile Ile Leu Ala Ile Ile Ile Ile Val Ile Leu
Ile Ile Ile Ile Val 85 90 95Ala Ile Val Gln Ser Gln Lys Lys
10021288PRTHomo sapiens 21Met Lys Asp Arg Thr Gln Glu Leu Arg Thr
Ala Lys Asp Ser Asp Asp 1 5 10 15Asp Asp Asp Val Ala Val Thr Val
Asp Arg Asp Arg Phe Met Asp Glu 20 25 30Phe Phe Glu Gln Val Glu Glu
Ile Arg Gly Phe Ile Asp Lys Ile Ala 35 40 45Glu Asn Val Glu Glu Val
Lys Arg Lys His Ser Ala Ile Leu Ala Ser 50 55 60Pro Asn Pro Asp Glu
Lys Thr Lys Glu Glu Leu Glu Glu Leu Met Ser65 70 75 80Asp Ile Lys
Lys Thr Ala Asn Lys Val Arg Ser Lys Leu Lys Ser Ile 85 90 95Glu Gln
Ser Ile Glu Gln Glu Glu Gly Leu Asn Arg Ser Ser Ala Asp 100 105
110Leu Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg Lys Phe Val
115 120 125Glu Val Met Ser Glu Tyr Asn Ala Thr Gln Ser Asp Tyr Arg
Glu Arg 130 135 140Cys Lys Gly Arg Ile Gln Arg Gln Leu Glu Ile Thr
Gly Arg Thr Thr145 150 155 160Thr Ser Glu Glu Leu Glu Asp Met Leu
Glu Ser Gly Asn Pro Ala Ile 165 170 175Phe Ala Ser Gly Ile Ile Met
Asp Ser Ser Ile Ser Lys Gln Ala Leu 180 185 190Ser Glu Ile Glu Thr
Arg His Ser Glu Ile Ile Lys Leu Glu Asn Ser 195 200 205Ile Arg Glu
Leu His Asp Met Phe Met Asp Met Ala Met Leu Val Glu 210 215 220Ser
Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu His Ala225 230
235 240Val Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala Val
Lys 245 250 255Tyr Gln Ser Lys Ala Arg Arg Lys Lys Ile Met Ile Ile
Ile Cys Cys 260 265 270Val Ile Leu Gly Ile Val Ile Ala Ser Thr Val
Gly Gly Ile Phe Ala 275 280 28522288PRTHomo sapiens 22Met Lys Asp
Arg Thr Gln Glu Leu Arg Ser Ala Lys Asp Ser Asp Asp 1 5 10 15Glu
Glu Glu Val Val His Val Asp Arg Asp His Phe Met Asp Glu Phe 20 25
30Phe Glu Gln Val Glu Glu Ile Arg Gly Cys Ile Glu Lys Leu Ser Glu
35 40 45Asp Val Glu Gln Val Lys Lys Gln His Ser Ala Ile Leu Ala Ala
Pro 50 55 60Asn Pro Asp Glu Lys Thr Lys Gln Glu Leu Glu Asp Leu Thr
Ala Asp65 70 75 80Ile Lys Lys Thr
Ala Asn Lys Val Arg Ser Lys Leu Lys Ala Ile Glu 85 90 95Gln Ser Ile
Glu Gln Glu Glu Gly Leu Asn Arg Ser Ser Ala Asp Leu 100 105 110Arg
Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg Lys Phe Val Glu 115 120
125Val Met Thr Glu Tyr Asn Ala Thr Gln Ser Lys Tyr Arg Asp Arg Cys
130 135 140Lys Asp Arg Ile Gln Arg Gln Leu Glu Ile Thr Gly Arg Thr
Thr Thr145 150 155 160Asn Glu Glu Leu Glu Asp Met Leu Glu Ser Gly
Lys Leu Ala Ile Phe 165 170 175Thr Asp Asp Ile Lys Met Asp Ser Gln
Met Thr Lys Gln Ala Leu Asn 180 185 190Glu Ile Glu Thr Arg His Asn
Glu Ile Ile Lys Leu Glu Thr Ser Ile 195 200 205Arg Glu Leu His Asp
Met Phe Val Asp Met Ala Met Leu Val Glu Ser 210 215 220Gln Gly Glu
Met Ile Asp Arg Ile Glu Tyr Asn Val Glu His Ser Val225 230 235
240Asp Tyr Val Glu Arg Ala Val Ser Asp Thr Lys Lys Ala Val Lys Tyr
245 250 255Gln Ser Lys Ala Arg Arg Lys Lys Ile Met Ile Ile Ile Cys
Cys Val 260 265 270Val Leu Gly Val Val Leu Ala Ser Ser Ile Gly Gly
Thr Leu Gly Leu 275 280 28523288PRTMus musculus 23Met Lys Asp Arg
Thr Gln Glu Leu Arg Thr Ala Lys Asp Ser Asp Asp 1 5 10 15Asp Asp
Asp Val Thr Val Thr Val Asp Arg Asp Arg Phe Met Asp Glu 20 25 30Phe
Phe Glu Gln Val Glu Glu Ile Arg Gly Phe Ile Asp Lys Ile Ala 35 40
45Glu Asn Val Glu Glu Val Lys Arg Lys His Ser Ala Ile Leu Ala Ser
50 55 60Pro Asn Pro Asp Glu Lys Thr Lys Glu Glu Leu Glu Glu Leu Met
Ser65 70 75 80Asp Ile Lys Lys Thr Ala Asn Lys Val Arg Ser Lys Leu
Lys Ser Ile 85 90 95Glu Gln Ser Ile Glu Gln Glu Glu Gly Leu Asn Arg
Ser Ser Ala Asp 100 105 110Leu Arg Ile Arg Lys Thr Gln His Ser Thr
Leu Ser Arg Lys Phe Val 115 120 125Glu Val Met Ser Glu Tyr Asn Ala
Thr Gln Ser Asp Tyr Arg Glu Arg 130 135 140Cys Lys Gly Arg Ile Gln
Arg Gln Leu Glu Ile Thr Gly Arg Thr Thr145 150 155 160Thr Ser Glu
Glu Leu Glu Asp Met Leu Glu Ser Gly Asn Pro Ala Ile 165 170 175Phe
Ala Ser Gly Ile Ile Met Asp Ser Ser Ile Ser Lys Gln Ala Leu 180 185
190Ser Glu Ile Glu Thr Arg His Ser Glu Ile Ile Lys Leu Glu Thr Ser
195 200 205Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu
Val Glu 210 215 220Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn
Val Glu His Ala225 230 235 240Val Asp Tyr Val Glu Arg Ala Val Ser
Asp Thr Lys Lys Ala Val Lys 245 250 255Tyr Gln Ser Lys Ala Arg Arg
Lys Lys Ile Met Ile Ile Ile Cys Cys 260 265 270Val Ile Leu Gly Ile
Ile Ile Ala Ser Thr Ile Gly Gly Ile Phe Gly 275 280
28524291PRTDrosophila sp. 24Met Thr Lys Asp Arg Leu Ala Ala Leu His
Ala Ala Gln Ser Asp Asp 1 5 10 15Glu Glu Glu Thr Glu Val Ala Val
Asn Val Asp Gly His Asp Ser Tyr 20 25 30Met Asp Asp Phe Phe Ala Gln
Val Glu Glu Ile Arg Gly Met Ile Asp 35 40 45Lys Val Gln Asp Asn Val
Glu Glu Val Lys Lys Lys His Ser Ala Ile 50 55 60Leu Ser Ala Pro Gln
Thr Asp Glu Lys Thr Lys Gln Glu Leu Glu Asp65 70 75 80Leu Met Ala
Asp Ile Lys Lys Asn Ala Asn Arg Val Arg Gly Lys Leu 85 90 95Lys Gly
Ile Glu Gln Asn Ile Glu Gln Glu Glu Gln Gln Asn Lys Ser 100 105
110Ser Ala Asp Leu Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg
115 120 125Lys Phe Val Glu Val Met Thr Glu Tyr Asn Arg Thr Gln Thr
Asp Tyr 130 135 140Arg Glu Arg Cys Lys Gly Arg Ile Gln Arg Gln Leu
Glu Ile Thr Gly145 150 155 160Arg Pro Thr Asn Asp Asp Glu Leu Glu
Lys Met Leu Glu Glu Gly Asn 165 170 175Ser Ser Val Phe Thr Gln Gly
Ile Ile Met Glu Thr Gln Gln Ala Lys 180 185 190Gln Thr Leu Ala Asp
Ile Glu Ala Arg His Gln Asp Ile Met Lys Leu 195 200 205Glu Thr Ser
Ile Lys Glu Leu His Asp Met Phe Met Asp Met Ala Met 210 215 220Leu
Val Glu Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr His Val225 230
235 240Glu His Ala Met Asp Tyr Val Gln Thr Ala Thr Gln Asp Thr Lys
Lys 245 250 255Ala Leu Lys Tyr Gln Ser Lys Ala Arg Arg Lys Lys Ile
Met Ile Leu 260 265 270Ile Cys Leu Thr Val Leu Gly Ile Leu Ala Ala
Ser Tyr Val Ser Ser 275 280 285Tyr Phe Met
29025291PRTCaenorhabditis elegans 25Met Thr Lys Asp Arg Leu Ser Ala
Leu Lys Ala Ala Gln Ser Glu Asp 1 5 10 15Glu Gln Asp Asp Asp Met
His Met Asp Thr Gly Asn Ala Gln Tyr Met 20 25 30Glu Glu Phe Phe Glu
Gln Val Glu Glu Ile Arg Gly Ser Val Asp Ile 35 40 45Ile Ala Asn Asn
Val Glu Glu Val Lys Lys Lys His Ser Ala Ile Leu 50 55 60Ser Asn Pro
Val Asn Asp Gln Lys Thr Lys Glu Glu Leu Asp Glu Leu65 70 75 80Met
Ala Val Ile Lys Arg Ala Ala Asn Lys Val Arg Gly Lys Leu Lys 85 90
95Leu Ile Glu Asn Ala Ile Asp His Asp Glu Gln Gly Ala Gly Asn Ala
100 105 110Asp Leu Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg
Arg Phe 115 120 125Val Glu Val Met Thr Asp Tyr Asn Lys Thr Gln Thr
Asp Tyr Arg Glu 130 135 140Arg Cys Lys Gly Arg Ile Gln Arg Gln Leu
Asp Ile Ala Gly Lys Gln145 150 155 160Val Gly Asp Glu Asp Leu Glu
Glu Met Ile Glu Ser Gly Asn Pro Gly 165 170 175Val Phe Thr Gln Gly
Ile Ile Thr Asp Thr Gln Gln Ala Lys Gln Thr 180 185 190Leu Ala Asp
Ile Glu Ala Arg His Asn Asp Ile Met Lys Leu Glu Ser 195 200 205Ser
Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu Val 210 215
220Glu Ser Gln Gly Glu Met Val Asp Arg Ile Glu Tyr Asn Val Glu
His225 230 235 240Ala Lys Glu Phe Val Asp Arg Ala Val Ala Asp Thr
Lys Lys Ala Val 245 250 255Gln Tyr Gln Ser Lys Ala Arg Arg Lys Lys
Ile Cys Ile Leu Val Thr 260 265 270Gly Val Ile Leu Ile Thr Gly Leu
Ile Ile Phe Ile Leu Phe Tyr Ala 275 280 285Lys Val Leu
29026288PRTStrongylocentrotus purpuratus 26Met Arg Asp Arg Leu Gly
Ser Leu Lys Arg Asn Glu Glu Asp Asp Val 1 5 10 15Gly Pro Glu Val
Ala Val Asn Val Glu Ser Glu Lys Phe Met Glu Glu 20 25 30Phe Phe Glu
Gln Val Glu Glu Val Arg Asn Asn Ile Asp Lys Ile Ser 35 40 45Lys Asn
Val Asp Glu Val Lys Lys Lys His Ser Asp Ile Leu Ser Ala 50 55 60Pro
Gln Ala Asp Glu Lys Val Lys Asp Glu Leu Glu Glu Leu Met Ser65 70 75
80Asp Ile Lys Lys Thr Ala Asn Lys Val Arg Ala Lys Leu Lys Met Met
85 90 95Glu Gln Ser Ile Glu Gln Glu Glu Ser Ala Lys Met Asn Ser Ala
Asp 100 105 110Val Arg Ile Arg Lys Thr Gln His Ser Thr Leu Ser Arg
Lys Phe Val 115 120 125Glu Val Met Thr Asp Tyr Asn Ser Thr Gln Thr
Asp Tyr Arg Glu Arg 130 135 140Cys Lys Gly Arg Ile Gln Arg Gln Leu
Glu Ile Thr Gly Lys Ser Thr145 150 155 160Thr Asp Ala Glu Leu Glu
Asp Met Leu Glu Ser Gly Asn Pro Ala Ile 165 170 175Phe Thr Ser Gly
Ile Ile Met Asp Thr Gln Gln Ala Lys Gln Thr Leu 180 185 190Arg Asp
Ile Glu Ala Arg His Asn Asp Ile Ile Lys Leu Glu Ser Ser 195 200
205Ile Arg Glu Leu His Asp Met Phe Met Asp Met Ala Met Leu Val Glu
210 215 220Ser Gln Gly Glu Met Ile Asp Arg Ile Glu Tyr Asn Val Glu
Gln Ser225 230 235 240Val Asp Tyr Val Glu Thr Ala Lys Met Asp Thr
Lys Lys Ala Val Lys 245 250 255Tyr Gln Ser Lys Ala Arg Arg Lys Lys
Phe Tyr Ile Ala Ile Cys Cys 260 265 270Gly Val Ala Leu Gly Ile Leu
Val Leu Val Leu Ile Ile Val Leu Ala 275 280 2852713PRTHomo sapiens
27Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met 1 5
102815PRTHomo sapiens 28Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln
Arg Ala Thr Lys 1 5 10 152916PRTHomo sapiens 29Ser Asn Lys Thr Arg
Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met 1 5 10 153017PRTHomo
sapiens 30Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr
Lys Met 1 5 10 15Leu3117PRTHomo sapiens 31Asp Ser Asn Lys Thr Arg
Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys 1 5 10 15Met3218PRTHomo
sapiens 32Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala
Thr Lys 1 5 10 15Met Leu3333PRTMus musculus 33Gln Asn Arg Gln Ile
Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys 1 5 10 15Thr Arg Ile
Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser 20 25 30Gly
3432PRTHomo sapiens 34Gln Asn Pro Gln Ile Lys Arg Ile Thr Asp Lys
Ala Asp Thr Asn Arg1 5 10 15Asp Arg Ile Asp Ile Ala Asn Ala Arg Ala
Lys Lys Leu Ile Asp Ser 20 25 303532PRTMus musculus 35Gln Asn Gln
Gln Ile Gln Lys Ile Thr Glu Lys Ala Asp Thr Asn Lys 1 5 10 15Asn
Arg Ile Asp Ile Ala Asn Thr Arg Ala Lys Lys Leu Ile Asp Ser 20 25
303634PRTGallus gallus 36Gln Asn Arg Gln Ile Asp Arg Ile Met Glu
Lys Leu Ile Pro Ile Lys 1 5 10 15Pro Gly Leu Met Lys Pro Thr Ser
Val Gln Gln Arg Cys Ser Ala Val 20 25 30Val Lys 3733PRTCarassius
auratus 37Gln Asn Arg Gln Ile Asp Arg Ile Met Asp Met Ala Asp Ser
Asn Lys 1 5 10 15Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys
Met Leu Gly Ser 20 25 30Gly3833PRTCarassius auratus 38Gln Asn Arg
Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys 1 5 10 15Thr
Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser 20 25
30Gly3930PRTTorpedo sp. 39Gln Asn Ala Gln Val Asp Arg Ile Val Val
Lys Gly Asp Met Asn Lys 1 5 10 15Ala Arg Ile Asp Glu Ala Asn Lys
His Ala Thr Lys Met Leu 20 25 304033PRTStrongylocentrotus
purpuratus 40Gln Asn Ser Gln Val Gly Arg Ile Thr Ser Lys Ala Glu
Ser Asn Glu 1 5 10 15Gly Arg Ile Asn Ser Ala Asp Lys Arg Ala Lys
Asn Ile Leu Arg Asn 20 25 30Lys4131PRTCaenorhabditis elagans 41Gln
Asn Arg Gln Leu Asp Arg Ile His Asp Lys Gln Ser Asn Glu Val 1 5 10
15Arg Val Glu Ser Ala Asn Lys Arg Ala Lys Asn Leu Ile Thr Lys 20 25
304231PRTDrosophila sp. 42Gln Asn Arg Gln Ile Asp Arg Ile Asn Arg
Lys Gly Glu Ser Asn Glu 1 5 10 15Ala Arg Ile Ala Val Ala Asn Gln
Arg Ala His Gln Leu Leu Lys 20 25 304332PRTHirudinida sp. 43Gln Asn
Arg Gln Val Asp Arg Ile Asn Asn Lys Met Thr Ser Asn Gln 1 5 10
15Leu Arg Ile Ser Asp Ala Asn Lys Arg Ala Ser Lys Leu Leu Lys Glu
20 25 304417PRTArtificial Sequencesynthetic peptide 44Ser Asn Lys
Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Ala 1 5 10
15Leu4517PRTArtificial Sequencesynthetic peptide 45Ser Asn Lys Thr
Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Xaa 1 5 10
15Leu4617PRTArtificial Sequencesynthetic peptide 46Ser Asn Lys Thr
Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Ala Met 1 5 10
15Leu4717PRTArtificial Sequencesynthetic peptide 47Ser Asn Lys Thr
Arg Ile Asp Glu Ala Asn Gln Arg Ala Ser Lys Met 1 5 10
15Leu4817PRTArtificial Sequencesynthetic peptide 48Ser Asn Lys Thr
Arg Ile Asp Glu Ala Asn Gln Arg Ala Xaa Lys Met 1 5 10
15Leu4917PRTArtificial Sequencesynthetic peptide 49Ser Asn Lys Thr
Arg Ile Asp Glu Ala Asn Gln Arg Xaa Thr Lys Met 1 5 10
15Leu5017PRTArtificial Sequencesynthetic peptide 50Ser Asn Lys Thr
Arg Ile Asp Glu Ala Asn Ala Arg Ala Thr Lys Met 1 5 10
15Leu5117PRTArtificial Sequencesynthetic peptide 51Ser Asn Lys Thr
Arg Ile Asp Glu Ala Asn Xaa Arg Ala Thr Lys Met 1 5 10
15Leu5217PRTArtificial Sequencesynthetic peptide 52Ser Asn Lys Thr
Arg Ile Asp Glu Ala Asn Asn Arg Ala Thr Lys Met 1 5 10
15Leu5317PRTArtificial Sequencesynthetic peptide 53Ser Asn Lys Thr
Arg Ile Asp Glu Ala Ala Gln Arg Ala Thr Lys Met 1 5 10
15Leu5417PRTArtificial Sequencesynthetic peptide 54Ser Asn Lys Thr
Arg Ile Asp Glu Xaa Asn Gln Arg Ala Thr Lys Met 1 5 10
15Leu5517PRTArtificial Sequencesynthetic peptide 55Ser Asn Lys Thr
Arg Ile Asp Gln Ala Asn Gln Arg Ala Thr Lys Met 1 5 10
15Leu5617PRTArtificial Sequencesynthetic peptide 56Ser Asn Lys Thr
Arg Ile Asn Glu Ala Asn Gln Arg Ala Thr Lys Met 1 5 10
15Leu5740PRTHomo sapiens 57Asp Lys Val Leu Glu Arg Asp Gln Lys Leu
Ser Glu Leu Asp Asp Arg 1 5 10 15Ala Asp Ala Leu Gln Ala Gly Ala
Ser Gln Phe Glu Ser Ser Ala Ala 20 25 30Lys Leu Lys Arg Lys Tyr Trp
Trp 35 405840PRTBos taurus 58Asp Lys Val Leu Glu Arg Asp Gln Lys
Leu Ser Glu Leu Asp Asp Arg1 5 10 15Ala Asp Ala Leu Gln Ala Gly Ala
Ser Gln Phe Glu Thr Ser Ala Ala 20 25 30Lys Leu Lys Arg Lys Tyr Trp
Trp 35 405940PRTRattus sp. 59Asp Lys Val Leu Glu Arg Asp Gln Lys
Leu Ser Glu Leu Asp Asp Arg 1 5 10 15Ala Asp Ala Leu Gln Ala Gly
Ala Ser Val Phe Glu Ser Ser Ala Ala 20 25 30Lys Leu Lys Arg Lys Tyr
Trp Trp 35 406040PRTRattus sp. 60Asp Lys Val Leu Glu Arg Asp Gln
Lys Leu Ser Glu Leu Asp Asp Arg 1 5 10 15Ala Asp Ala Leu Gln Ala
Gly Ala Ser Gln Phe Glu Thr Ser Ala Ala 20 25 30Lys Leu Lys Arg Lys
Tyr Trp Trp 35 406140PRTRattus sp. 61Asp Lys Val Leu Glu Arg Asp
Gln Lys Leu Ser Glu Leu Asp Asp Arg 1 5 10 15Ala Asp Ala Leu Gln
Ala Gly Ala Ser Gln Phe Glu Thr Ser Ala Ala 20 25 30Lys Leu Lys Arg
Lys Tyr Trp Trp 35 406240PRTRattus sp. 62Asp Leu Val Ala Gln Arg
Gly Glu Arg Leu Glu Leu Leu Ile Asp Lys 1 5 10 15Thr Glu Asn Leu
Val Asp Ser Ser Val Thr Phe Lys Thr Thr Ser Arg 20 25 30Asn Leu Ala
Arg Ala Met Cys Met 35 406332PRTGallus gallus 63Glu Arg Asp Gln Lys
Leu Ser Glu Leu Asp Asp Arg Ala Asp Ala Leu 1 5 10 15Gln Ala Gly
Ala Ser Val Phe Glu Ser Ser Ala Ala Lys Leu Lys Arg
20 25 306432PRTGallus gallus 64Glu Arg Asp Gln Lys Leu Ser Glu Leu
Asp Asp Arg Ala Asp Ala Leu 1 5 10 15Gln Ala Gly Ala Ser Gln Phe
Glu Thr Ser Ala Ala Lys Leu Lys Arg 20 25 306540PRTTorpedo sp.
65Asp Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu Asp Asp Arg 1
5 10 15Ala Asp Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Ser Ser Ala
Ala 20 25 30Lys Leu Lys Arg Lys Tyr Trp Trp 35
406640PRTStrongylocentrotus purpuratus 66Asp Lys Val Leu Asp Arg
Asp Gly Ala Leu Ser Val Leu Asp Asp Arg 1 5 10 15Ala Asp Ala Leu
Gln Gln Gly Ala Ser Gln Phe Glu Thr Asn Ala Gly 20 25 30Lys Leu Lys
Arg Lys Tyr Trp Trp 35 406740PRTAplysia sp. 67Glu Lys Val Leu Asp
Arg Asp Gln Lys Ile Ser Gln Leu Asp Asp Arg 1 5 10 15Ala Glu Ala
Leu Gln Ala Gly Ala Ser Gln Phe Glu Ala Ser Ala Gly 20 25 30Lys Leu
Lys Arg Lys Tyr Trp Trp 35 406840PRTTeuthoida sp. 68Asp Lys Val Leu
Glu Arg Asp Ser Lys Ile Ser Glu Leu Asp Asp Arg 1 5 10 15Ala Asp
Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu Ala Ser Ala Gly 20 25 30Lys
Leu Lys Arg Lys Phe Trp Trp 35 406940PRTCaenorhabditis elegans
69Asn Lys Val Met Glu Arg Asp Val Gln Leu Asn Ser Leu Asp His Arg 1
5 10 15Ala Glu Val Leu Gln Asn Gly Ala Ser Gln Phe Gln Gln Ser Ser
Arg 20 25 30Glu Leu Lys Arg Gln Tyr Trp Trp 35 407040PRTDrosophila
sp. 70Glu Lys Val Leu Glu Arg Asp Gln Lys Leu Ser Glu Leu Gly Glu
Arg 1 5 10 15Ala Asp Gln Leu Glu Gly Gly Ala Ser Gln Ser Glu Gln
Gln Ala Gly 20 25 30Lys Leu Lys Arg Lys Gln Trp Trp 35
407140PRTDrosophila sp. 71Glu Lys Val Leu Glu Arg Asp Ser Lys Leu
Ser Glu Leu Asp Asp Arg 1 5 10 15Ala Asp Ala Leu Gln Gln Gly Ala
Ser Gln Phe Glu Gln Gln Ala Gly 20 25 30Lys Leu Lys Arg Lys Phe Trp
Leu 35 407239PRTHirudinida sp. 72Asp Lys Val Leu Glu Lys Asp Gln
Lys Leu Ala Glu Leu Asp Arg Ala 1 5 10 15Asp Ala Leu Gln Ala Gly
Ala Ser Gln Phe Glu Ala Ser Ala Gly Lys 20 25 30Leu Lys Arg Lys Phe
Trp Trp 357318PRTHomo sapiens 73Glu Arg Ala Val Ser Asp Thr Lys Lys
Ala Val Lys Tyr Gln Ser Lys 1 5 10 15Ala Arg7418PRTBos taurus 74Glu
Arg Ala Val Ser Asp Thr Lys Lys Ala Val Lys Tyr Gln Ser Lys 1 5 10
15Ala Arg7518PRTRattus sp. 75Glu His Ala Lys Glu Glu Thr Lys Lys
Ala Ile Lys Tyr Gln Ser Lys 1 5 10 15Ala Arg7618PRTRattus sp. 76Glu
Lys Ala Arg Asp Glu Thr Arg Lys Ala Met Lys Tyr Gln Gly Gly 1 5 10
15Ala Arg7718PRTRattus sp. 77Glu Arg Gly Gln Glu His Val Lys Ile
Ala Leu Glu Asn Gln Lys Lys 1 5 10 15Ala Arg7818PRTGallus gallus
78Val Pro Glu Val Phe Val Thr Lys Ser Ala Val Met Tyr Gln Cys Lys 1
5 10 15Ser Arg7918PRTStrongylocentrotus purpuratus 79Val Arg Arg
Gln Asn Asp Thr Lys Lys Ala Val Lys Tyr Gln Ser Lys 1 5 10 15Ala
Arg8018PRTAplysia sp. 80Glu Thr Ala Lys Met Asp Thr Lys Lys Ala Val
Lys Tyr Gln Ser Lys 1 5 10 15Ala Arg8118PRTTeuthoida sp. 81Glu Thr
Ala Lys Val Asp Thr Lys Lys Ala Val Lys Tyr Gln Ser Lys 1 5 10
15Ala Arg8218PRTDrosophila sp. 82Gln Thr Ala Thr Gln Asp Thr Lys
Lys Ala Leu Lys Tyr Gln Ser Lys 1 5 10 15Ala Arg8318PRTHirudinida
sp. 83Glu Thr Ala Ala Ala Asp Thr Lys Lys Ala Met Lys Tyr Gln Ser
Ala 1 5 10 15Ala Arg845PRTArtificial Sequencesynthetic construct
84Gly Gly Gly Gly Ser 1 5851002DNAArtificial Sequencesynthetic
construct 85atg gct agc aaa gga gaa gaa ctc ttc act gga gtt gtc cca
att ctt 48Met Ala Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro
Ile Leu1 5 10 15gtt gaa tta gat ggt gat gtt aac ggc cac aag ttc tct
gtc agt gga 96Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser
Val Ser Gly 20 25 30gag ggt gaa ggt gat gca aca tac gga aaa ctt acc
ctg aag ttc atc 144Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr
Leu Lys Phe Ile 35 40 45tgc act act ggc aaa ctg cct gtt cca tgg cca
aca cta gtc act act 192Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro
Thr Leu Val Thr Thr 50 55 60ctg tgc tat ggt gtt caa tgc ttt tca aga
tac ccg gat cat atg aaa 240Leu Cys Tyr Gly Val Gln Cys Phe Ser Arg
Tyr Pro Asp His Met Lys65 70 75 80cgg cat gac ttt ttc aag agt gcc
atg ccc gaa ggt tat gta cag gaa 288Arg His Asp Phe Phe Lys Ser Ala
Met Pro Glu Gly Tyr Val Gln Glu 85 90 95agg acc atc ttc ttc aaa gat
gac ggc aac tac aag aca cgt gct gaa 336Arg Thr Ile Phe Phe Lys Asp
Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110gtc aag ttt gaa ggt
gat acc ctt gtt aat aga atc gag tta aaa ggt 384Val Lys Phe Glu Gly
Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125att gac ttc
aag gaa gat ggc aac att ctg gga cac aaa ttg gaa tac 432Ile Asp Phe
Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140aac
tat aac tca cac aat gta tac atc atg gca gac aaa caa aag aat 480Asn
Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150
155 160gga atc aaa gtg aac ttc aag acc cgc cac aac att gaa gat gga
agc 528Gly Ile Lys Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly
Ser 165 170 175gtt caa cta gca gac cat tat caa caa aat act cca att
ggc gat ggc 576Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile
Gly Asp Gly 180 185 190cct gtc ctt tta cca gac aac cat tac ctg tcc
aca caa tct gcc ctt 624Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser
Thr Gln Ser Ala Leu 195 200 205tcg aaa gat ccc aac gaa aag aga gac
cac atg gtc ctt ctt gag ttt 672Ser Lys Asp Pro Asn Glu Lys Arg Asp
His Met Val Leu Leu Glu Phe 210 215 220gta aca gct gct ggg att aca
cat ggc atg gat gaa ctg tac aac ggc 720Val Thr Ala Ala Gly Ile Thr
His Gly Met Asp Glu Leu Tyr Asn Gly225 230 235 240ggt gca gga tcc
ggt gcg ggt ggc ggt ggc atc cgg agg gta aca aac 768Gly Ala Gly Ser
Gly Ala Gly Gly Gly Gly Ile Arg Arg Val Thr Asn 245 250 255gat gcc
cgg gaa aat gag atg gat gag aac ctg gag cag gtg agc ggc 816Asp Ala
Arg Glu Asn Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly 260 265
270atc atc gga aac ctc cgc cat atg gct cta gac atg ggc aat gag att
864Ile Ile Gly Asn Leu Arg His Met Ala Leu Asp Met Gly Asn Glu Ile
275 280 285gac acc cag aat cgc cag atc gac agg atc atg gag aag gct
gat tcc 912Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala
Asp Ser 290 295 300aac aaa acc aga att gat gaa gcc aac caa cgt gca
aca aag atg ctg 960Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala
Thr Lys Met Leu305 310 315 320gga agt ggt ggc ggt ggc ggc cat cac
cat cac cat cac taa 1002Gly Ser Gly Gly Gly Gly Gly His His His His
His His * 325 33086333PRTArtificial Sequencesynthetic construct
86Met Ala Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1
5 10 15Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser
Gly 20 25 30Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys
Phe Ile 35 40 45Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu
Val Thr Thr 50 55 60Leu Cys Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro
Asp His Met Lys65 70 75 80Arg His Asp Phe Phe Lys Ser Ala Met Pro
Glu Gly Tyr Val Gln Glu 85 90 95Arg Thr Ile Phe Phe Lys Asp Asp Gly
Asn Tyr Lys Thr Arg Ala Glu 100 105 110Val Lys Phe Glu Gly Asp Thr
Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125Ile Asp Phe Lys Glu
Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140Asn Tyr Asn
Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn145 150 155
160Gly Ile Lys Val Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser
165 170 175Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly
Asp Gly 180 185 190Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr
Gln Ser Ala Leu 195 200 205Ser Lys Asp Pro Asn Glu Lys Arg Asp His
Met Val Leu Leu Glu Phe 210 215 220Val Thr Ala Ala Gly Ile Thr His
Gly Met Asp Glu Leu Tyr Asn Gly225 230 235 240Gly Ala Gly Ser Gly
Ala Gly Gly Gly Gly Ile Arg Arg Val Thr Asn 245 250 255Asp Ala Arg
Glu Asn Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly 260 265 270Ile
Ile Gly Asn Leu Arg His Met Ala Leu Asp Met Gly Asn Glu Ile 275 280
285Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser
290 295 300Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys
Met Leu305 310 315 320Gly Ser Gly Gly Gly Gly Gly His His His His
His His 325 330871044DNAArtificial Sequencesynthetic construct
87atg gct agc gga gga ctg aac gac atc ttc gag gct caa aag atc gag
48Met Ala Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu1
5 10 15tgg cat gga tcc cat cat cat cat cat cat cat cat atc cgg agg
gta 96Trp His Gly Ser His His His His His His His His Ile Arg Arg
Val 20 25 30aca aac gat gcc cgg gaa aat gag atg gat gag aac ctg gag
cag gtg 144Thr Asn Asp Ala Arg Glu Asn Glu Met Asp Glu Asn Leu Glu
Gln Val 35 40 45agc ggc atc atc gga aac ctc cgc cat atg gct cta gac
atg ggc aat 192Ser Gly Ile Ile Gly Asn Leu Arg His Met Ala Leu Asp
Met Gly Asn 50 55 60gag att gac acc cag aat cgc cag atc gac agg atc
atg gag aag gct 240Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile
Met Glu Lys Ala65 70 75 80gat tcc aac aaa acc aga att gat gaa gcc
aac caa cgt gca aca aag 288Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala
Asn Gln Arg Ala Thr Lys 85 90 95atg ctg gga agt ggt ggc ggt ggt agc
ggc acc ggt ggc gct agc aaa 336Met Leu Gly Ser Gly Gly Gly Gly Ser
Gly Thr Gly Gly Ala Ser Lys 100 105 110gga gaa gaa ctc ttc act gga
gtt gtc cca att ctt gtt gaa tta gat 384Gly Glu Glu Leu Phe Thr Gly
Val Val Pro Ile Leu Val Glu Leu Asp 115 120 125ggt gat gtt aac ggc
cac aag ttc tct gtc agt gga gag ggt gaa ggt 432Gly Asp Val Asn Gly
His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly 130 135 140gat gca aca
tac gga aaa ctt acc ctg aag ttc atc tgc act act ggc 480Asp Ala Thr
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly145 150 155
160aaa ctg cct gtt cca tgg cca aca cta gtc act act ctg tgc tat ggt
528Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Cys Tyr Gly
165 170 175gtt caa tgc ttt tca aga tac ccg gat cat atg aaa cgg cat
gac ttt 576Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His
Asp Phe 180 185 190ttc aag agt gcc atg ccc gaa ggt tat gta cag gaa
agg acc atc ttc 624Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
Arg Thr Ile Phe 195 200 205ttc aaa gat gac ggc aac tac aag aca cgt
gct gaa gtc aag ttt gaa 672Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg
Ala Glu Val Lys Phe Glu 210 215 220ggt gat acc ctt gtt aat aga atc
gag tta aaa ggt att gac ttc aag 720Gly Asp Thr Leu Val Asn Arg Ile
Glu Leu Lys Gly Ile Asp Phe Lys225 230 235 240gaa gat ggc aac att
ctg gga cac aaa ttg gaa tac aac tat aac tca 768Glu Asp Gly Asn Ile
Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser 245 250 255cac aat gta
tac atc atg gca gac aaa caa aag aat gga atc aaa gtg 816His Asn Val
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val 260 265 270aac
ttc aag acc cgc cac aac att gaa gat gga agc gtt caa cta gca 864Asn
Phe Lys Thr Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala 275 280
285gac cat tat caa caa aat act cca att ggc gat ggc cct gtc ctt tta
912Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu
290 295 300cca gac aac cat tac ctg tcc aca caa tct gcc ctt tcg aaa
gat ccc 960Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys
Asp Pro305 310 315 320aac gaa aag aga gac cac atg gtc ctt ctt gag
ttt gta aca gct gct 1008Asn Glu Lys Arg Asp His Met Val Leu Leu Glu
Phe Val Thr Ala Ala 325 330 335ggg att aca cat ggc atg gat gaa ctg
tac aac tga 1044Gly Ile Thr His Gly Met Asp Glu Leu Tyr Asn * 340
34588347PRTArtificial Sequencesynthetic construct 88Met Ala Ser Gly
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu 1 5 10 15Trp His
Gly Ser His His His His His His His His Ile Arg Arg Val 20 25 30Thr
Asn Asp Ala Arg Glu Asn Glu Met Asp Glu Asn Leu Glu Gln Val 35 40
45Ser Gly Ile Ile Gly Asn Leu Arg His Met Ala Leu Asp Met Gly Asn
50 55 60Glu Ile Asp Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys
Ala65 70 75 80Asp Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg
Ala Thr Lys 85 90 95Met Leu Gly Ser Gly Gly Gly Gly Ser Gly Thr Gly
Gly Ala Ser Lys 100 105 110Gly Glu Glu Leu Phe Thr Gly Val Val Pro
Ile Leu Val Glu Leu Asp 115 120 125Gly Asp Val Asn Gly His Lys Phe
Ser Val Ser Gly Glu Gly Glu Gly 130 135 140Asp Ala Thr Tyr Gly Lys
Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly145 150 155 160Lys Leu Pro
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Cys Tyr Gly 165 170 175Val
Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Arg His Asp Phe 180 185
190Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe
195 200 205Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys
Phe Glu 210 215 220Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
Ile Asp Phe Lys225 230 235 240Glu Asp Gly Asn Ile Leu Gly His Lys
Leu Glu Tyr Asn Tyr Asn Ser 245 250 255His Asn Val Tyr Ile Met Ala
Asp Lys Gln Lys Asn Gly Ile Lys Val 260 265 270Asn Phe Lys Thr Arg
His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala 275 280 285Asp His Tyr
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu 290 295 300Pro
Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro305 310
315 320Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala
Ala 325 330 335Gly Ile Thr His Gly Met Asp Glu Leu Tyr Asn 340
345892040DNAArtificial Sequencesynthetic construct 89cccgaggttt
ggagctgtct ttccttccct ccctacccgg cggctcctcc actcttgcta 60cctgcaggga
tcagcggaca gcatcctctg aagaagacaa ggttccttaa ctaagcacca
120ctgacttgct ggccccggcg cccagcaacc ccccaccact acc atg gcc gag gac
175
Met Ala Glu Asp 1gca gac atg cgt aat gaa ctg gag gag atg cag agg
agg gct gac cag 223Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg
Arg Ala Asp Gln5 10 15 20ctg gct gat gag tcc ctg gaa agc acc cgt
cgc atg ctg cag ctg gtc 271Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg
Arg Met Leu Gln Leu Val 25 30 35gaa gag agt aaa gat gct ggc atc agg
act ttg gtt atg ttg gat gag 319Glu Glu Ser Lys Asp Ala Gly Ile Arg
Thr Leu Val Met Leu Asp Glu 40 45 50caa ggc gaa caa ctg gaa cgc att
gag gaa ggg atg gac caa atc aat 367Gln Gly Glu Gln Leu Glu Arg Ile
Glu Glu Gly Met Asp Gln Ile Asn 55 60 65aag gat atg aaa gaa gca gaa
aag aat ttg acg gac cta gga aaa ttc 415Lys Asp Met Lys Glu Ala Glu
Lys Asn Leu Thr Asp Leu Gly Lys Phe 70 75 80tgc ggg ctt tgt gtg tgt
ccc tgt aac aag ctt aaa tcc agt gat gct 463Cys Gly Leu Cys Val Cys
Pro Cys Asn Lys Leu Lys Ser Ser Asp Ala85 90 95 100tac aaa aaa gcc
tgg ggc aat aat cag gat gga gta gtg gcc agc cag 511Tyr Lys Lys Ala
Trp Gly Asn Asn Gln Asp Gly Val Val Ala Ser Gln 105 110 115cct gcc
cgt gtg gtg gat gaa cgg gag cag atg gcc atc agt ggt ggc 559Pro Ala
Arg Val Val Asp Glu Arg Glu Gln Met Ala Ile Ser Gly Gly 120 125
130ttc atc cgc agg gta aca aac gat gcc cgg gaa aat gaa atg gat gaa
607Phe Ile Arg Arg Val Thr Asn Asp Ala Arg Glu Asn Glu Met Asp Glu
135 140 145aac cta gag cag gtg agc ggc atc atc gga aac ctc cgt cat
atg gcc 655Asn Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu Arg His
Met Ala 150 155 160cta gac atg ggc aat gag att gac acc cag aat cgc
cag att gac agg 703Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg
Gln Ile Asp Arg165 170 175 180atc atg gag aag gct gac tcc aac aaa
acc aga att gat gaa gcc aac 751Ile Met Glu Lys Ala Asp Ser Asn Lys
Thr Arg Ile Asp Glu Ala Asn 185 190 195caa cgt gca aca aag atg ctg
gga agt ggt taa atctgccgtt ctgctgtgct 804Gln Arg Ala Thr Lys Met
Leu Gly Ser Gly * 200 205gtcctccaat gttgttggac aagagagaag
agagctcctt catgcttctc tcatggtatt 864acctagtaag acttacacac
acacacacac acacacacac acacacacac acacacacac 924acacacagag
tagtcacccc cattgtaaat gtctgtgtgg tttgtcagct tcccaatgat
984accatgtgtc ttttgttttc tccggctctc tttctttgcc aaaggttgta
catagtggtc 1044atctggtgac tctatttcct gacttaagag ttcttgggtc
tctctctttc ttttctcagt 1104ggcgtttgct gaatgacaac aatttaggaa
tgctcaatgt actgttgatt tttctcaata 1164cacagtattg ttcttgtaaa
actgtgactt accacagagc tactaccaca gtcctttctt 1224agggtgtcag
gctctgaatc tctccaaatg tgctctcttt ggttcctcag tgctattctt
1284tgtctttatg atttcataat tagacaatgt gaaattacat aacaggcatt
gcactaaaag 1344tgatgtgatt tatgcattta tgcatgagaa ctaaatagac
ttttagatcc tacttaaaca 1404aaaacttcca tgacagtagc atactgacaa
gaaaacacac acaacagcaa caataacaaa 1464gcaacaacta cgcatgctca
gcattgggac actgtcaaga ttaagtcata ccagcaaaac 1524ctgcagctgt
gtcaccttct tctgtcaaca tacagactga tcataatgat cccttcttta
1584cacacacaca cacacacaca cacacacaca cacacacaaa tggaatttaa
ccaacttccc 1644agaattgatg aagcaaatat atgtttggct gaaactattg
taaatgggtg taatataggg 1704tttgtcgaat gcttttgaaa gctctgtttt
ccagacaata ctcttgtgtg gaaaacgtga 1764agatcttcta agtctggctc
ttgtgatcac caaaccctgg tgcatcagta caacactttg 1824cgctaatcta
gagctatgca caaccaaatt gctgagatgt ttagtagctg ataaagaaac
1884ctttaaaaaa ttatataaat gaatgaaata tagataaact gtgagataaa
tatcattaca 1944gcatgtatat taaatccctc ctgtctcctc tgttggtttg
tgaagtgatt tgacattttg 2004tagctagttt aaaattatta aaaattatag atgtta
204090206PRTArtificial Sequencesynthetic construct 90Met Ala Glu
Asp Ala Asp Met Arg Asn Glu Leu Glu Glu Met Gln Arg 1 5 10 15Arg
Ala Asp Gln Leu Ala Asp Glu Ser Leu Glu Ser Thr Arg Arg Met 20 25
30Leu Gln Leu Val Glu Glu Ser Lys Asp Ala Gly Ile Arg Thr Leu Val
35 40 45Met Leu Asp Glu Gln Gly Glu Gln Leu Glu Arg Ile Glu Glu Gly
Met 50 55 60Asp Gln Ile Asn Lys Asp Met Lys Glu Ala Glu Lys Asn Leu
Thr Asp65 70 75 80Leu Gly Lys Phe Cys Gly Leu Cys Val Cys Pro Cys
Asn Lys Leu Lys 85 90 95Ser Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn
Asn Gln Asp Gly Val 100 105 110Val Ala Ser Gln Pro Ala Arg Val Val
Asp Glu Arg Glu Gln Met Ala 115 120 125Ile Ser Gly Gly Phe Ile Arg
Arg Val Thr Asn Asp Ala Arg Glu Asn 130 135 140Glu Met Asp Glu Asn
Leu Glu Gln Val Ser Gly Ile Ile Gly Asn Leu145 150 155 160Arg His
Met Ala Leu Asp Met Gly Asn Glu Ile Asp Thr Gln Asn Arg 165 170
175Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn Lys Thr Arg Ile
180 185 190Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly Ser Gly
195 200 205918PRTArtificial Sequencesynthetic construct 91Asp Tyr
Lys Asp Asp Asp Asp Lys 1 5929PRTArtificial Sequencesynthetic
construct 92Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1
59310PRTArtificial Sequencesynthetic construct 93Glu Gln Lys Leu
Ile Ser Glu Glu Asp Leu 1 5 10946PRTArtificial Sequencesynthetic
construct 94Asp Thr Tyr Arg Tyr Ile 1 5956PRTArtificial
Sequencesynthetic construct 95His His His His His His 1
596118PRTHomo sapiens 96Met Ser Ala Pro Ala Gln Pro Pro Ala Glu Gly
Thr Glu Gly Thr Ala 1 5 10 15Pro Gly Gly Gly Pro Pro Gly Pro Pro
Pro Asn Met Thr Ser Asn Arg 20 25 30Arg Leu Gln Gln Thr Gln Ala Gln
Val Glu Glu Val Val Asp Ile Ile 35 40 45Arg Val Asn Val Asp Lys Val
Leu Glu Arg Asp Gln Lys Leu Ser Glu 50 55 60Leu Asp Asp Arg Ala Asp
Ala Leu Gln Ala Gly Ala Ser Gln Phe Glu65 70 75 80Ser Ser Ala Ala
Lys Leu Lys Arg Lys Tyr Trp Trp Lys Asn Cys Lys 85 90 95Met Met Ile
Met Leu Gly Ala Ile Cys Ala Ile Ile Val Val Val Ile 100 105 110Val
Ile Tyr Phe Phe Thr 11597714DNAAequorea victoriaCDS(1)...(714)
97atg agt aaa gga gaa gaa ctt ttc act gga gtt gtc cca att ctt gtt
48Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val1
5 10 15gaa tta gat ggt gat gtt aat ggg caa aaa ttc tct gtc agg gga
gag 96Glu Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Arg Gly
Glu 20 25 30ggt gaa ggt gat gca aca tac gga aaa ctt acc ctt aaa ttt
att tgc 144Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe
Ile Cys 35 40 45act act ggg aag cta cct gtt cca tgg cca aca ctt gtc
act act ttc 192Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val
Thr Thr Phe 50 55 60tct tat ggt gta caa tgc ttc tca aga tac cca gat
cat atg aaa cag 240Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp
His Met Lys Gln65 70 75 80cat gac ttt ctc aag agt gcc atg ccc gaa
ggt tat gta cag gaa aga 288His Asp Phe Leu Lys Ser Ala Met Pro Glu
Gly Tyr Val Gln Glu Arg 85 90 95act ata ttt tac aaa gat gac ggg aac
tac aag aca cgt gct gaa gtc 336Thr Ile Phe Tyr Lys Asp Asp Gly Asn
Tyr Lys Thr Arg Ala Glu Val 100 105 110aag ttt gag ggt gat acc ctt
gtt aat aga atc gag tta aaa ggt att 384Lys Phe Glu Gly Asp Thr Leu
Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125gat ttt aaa gaa gat
gga aac att ctt gga cac aaa atg gaa tac aac 432Asp Phe Lys Glu Asp
Gly Asn Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140tat aac tca
cat aat gta tac atc atg gga gac aaa cca aag aat ggc 480Tyr Asn Ser
His Asn Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly145 150 155
160atc aaa gtt aac ttc aaa att aga cac aac att aaa gat gga agc gtt
528Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val
165 170 175caa tta gca gac cat tat caa caa aat act cca att ggc gat
ggc cct 576Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp
Gly Pro 180 185 190gtc ctt tta cca gac aac cat tac ctg tcc aca caa
tct gcc ctt tcc 624Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln
Ser Ala Leu Ser 195 200 205caa gat ccc cac gga aag aga gat cac atg
gtc ctt ctt gag ttt gtt 672Gln Asp Pro His Gly Lys Arg Asp His Met
Val Leu Leu Glu Phe Val 210 215 220aca tct gct ggg att aca cat ggc
atg gat gaa cta tac aaa 714Thr Ser Ala Gly Ile Thr His Gly Met Asp
Glu Leu Tyr Lys225 230 23598238PRTAequorea victoria 98Met Ser Lys
Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15Glu
Leu Asp Gly Asp Val Asn Gly Gln Lys Phe Ser Val Arg Gly Glu 20 25
30Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
35 40 45Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
Phe 50 55 60Ser Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met
Lys Gln65 70 75 80His Asp Phe Leu Lys Ser Ala Met Pro Glu Gly Tyr
Val Gln Glu Arg 85 90 95Thr Ile Phe Tyr Lys Asp Asp Gly Asn Tyr Lys
Thr Arg Ala Glu Val 100 105 110Lys Phe Glu Gly Asp Thr Leu Val Asn
Arg Ile Glu Leu Lys Gly Ile 115 120 125Asp Phe Lys Glu Asp Gly Asn
Ile Leu Gly His Lys Met Glu Tyr Asn 130 135 140Tyr Asn Ser His Asn
Val Tyr Ile Met Gly Asp Lys Pro Lys Asn Gly145 150 155 160Ile Lys
Val Asn Phe Lys Ile Arg His Asn Ile Lys Asp Gly Ser Val 165 170
175Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
180 185 190Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala
Leu Ser 195 200 205Gln Asp Pro His Gly Lys Arg Asp His Met Val Leu
Leu Glu Phe Val 210 215 220Thr Ser Ala Gly Ile Thr His Gly Met Asp
Glu Leu Tyr Lys225 230 235996PRTArtificial Sequencesynthetic
construct 99Cys Cys Xaa Xaa Cys Cys 1 51006PRTArtificial
Sequencesynthetic construct 100Cys Cys Pro Gly Cys Cys 1
510124DNAArtificial Sequenceprimer 101atccggaggg taacaaacga tgcc
2410260DNAArtificial Sequenceprimer 102cgaattccgc gggccaccat
gggaggagga ctgaacgaca tcttcgaggc tcaaaagatc 6010379DNAArtificial
Sequenceprimer 103tcgtttgtta ccctccggat atgatgatga tgatgatgat
gatgggatcc atgccactcg 60atcttttgag cctcgaaga 7910436DNAArtificial
Sequenceprimer 104gctagatctc gagttaacca cttcccagca tctttg
3610530DNAArtificial Sequenceprimer 105cgaagatctg gaggactgaa
cgacatcttc 3010633DNAArtificial Sequencesynthetic construct
106gatgaagcca accaagctgc aacaaagatg ctg 3310731DNAArtificial
Sequencesynthetic construct 107cgccagatcg acgatatcat ggagaaggct g
3110810PRTArtificial Sequencesynthetic construct 108His His His His
His His His His His His 1 5 1010918PRTClostridium sp. 109Ser Asn
Arg Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Arg Met 1 5 10
15Leu Gly11035PRTClostridium sp. 110Leu Ser Glu Leu Asp Asp Arg Ala
Asp Ala Leu Gln Ala Gly Ala Ser 1 5 10 15Gln Phe Glu Thr Ser Ala
Ala Lys Leu Lys Arg Lys Tyr Trp Trp Lys 20 25 30Asn Leu Lys
3511139PRTClostridium sp. 111Ala Gln Val Asp Glu Val Val Asp Ile
Met Arg Val Asn Val Asp Lys 1 5 10 15Val Leu Glu Arg Asp Gln Lys
Leu Ser Glu Leu Asp Asp Arg Ala Asp 20 25 30Ala Leu Gln Ala Gly Ala
Ser 35112114PRTClostridium sp. 112Asn Lys Leu Lys Ser Ser Asp Ala
Tyr Lys Lys Ala Trp Gly Asn Asn 1 5 10 15Gln Asp Gly Val Val Ala
Ser Gln Pro Ala Arg Val Val Asp Glu Arg 20 25 30Glu Gln Met Ala Ile
Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp 35 40 45Ala Arg Glu Asn
Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile 50 55 60Ile Gly Asn
Leu Arg Gly Met Ala Leu Asp Met Gly Asn Glu Ile Asp65 70 75 80Thr
Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser Asn 85 90
95Lys Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys Met Leu Gly
100 105 110Ser Gly 113114PRTClostridium sp. 113Asn Lys Leu Lys Ser
Ser Asp Ala Tyr Lys Lys Ala Trp Gly Asn Asn 1 5 10 15Gln Asp Gly
Val Val Ala Ser Gln Pro Ala Arg Val Val Asp Glu Arg 20 25 30Glu Gln
Met Ala Ile Ser Gly Gly Phe Ile Arg Arg Val Thr Asn Asp 35 40 45Ala
Arg Glu Asn Glu Met Asp Glu Asn Leu Glu Gln Val Ser Gly Ile 50 55
60Ile Gly Asn Leu Arg His Met Ala Leu Asp Met Gly Asn Glu Ile Asp65
70 75 80Thr Gln Asn Arg Gln Ile Asp Arg Ile Met Glu Lys Ala Asp Ser
Asn 85 90 95Lys Thr Arg Ile Asp Glu Ala Asn Gln Ala Ala Thr Lys Met
Leu Gly 100 105 110Ser Gly
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