U.S. patent application number 12/153942 was filed with the patent office on 2009-07-16 for low-molecular weight peptides inhibiting ion channel activity.
This patent application is currently assigned to PHARMADESIGN, INC.. Invention is credited to Toshio Furuya, Masahiro Sokabe, Takane Yokotagawa.
Application Number | 20090181899 12/153942 |
Document ID | / |
Family ID | 33095036 |
Filed Date | 2009-07-16 |
United States Patent
Application |
20090181899 |
Kind Code |
A1 |
Yokotagawa; Takane ; et
al. |
July 16, 2009 |
Low-molecular weight peptides inhibiting ion channel activity
Abstract
It is intended to provide novel polypeptides which specifically
inhibit the activity of a mechano-sensitive channel; and
mechano-sensitive channel inhibitors or remedies for atrial
fibrillation containing these polypeptides or salts thereof. The
above objects can be achieved by using polypeptides having amino
acid sequences represented by SEQ ID NO: 1 (TVP003), SEQ ID NO:2
(TVP004) and SEQ ID NO:3 (TVP005), salts of these polypeptides, and
mechano-sensitive channel inhibitors or remedies for atrial
fibrillation containing the same.
Inventors: |
Yokotagawa; Takane; (Tokyo,
JP) ; Sokabe; Masahiro; (Aichi, JP) ; Furuya;
Toshio; (Tokyo, JP) |
Correspondence
Address: |
WESTERMAN, HATTORI, DANIELS & ADRIAN, LLP
1250 CONNECTICUT AVENUE, NW, SUITE 700
WASHINGTON
DC
20036
US
|
Assignee: |
PHARMADESIGN, INC.
Tokyo
JP
|
Family ID: |
33095036 |
Appl. No.: |
12/153942 |
Filed: |
May 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10550102 |
Sep 21, 2005 |
7396816 |
|
|
PCT/JP2004/004190 |
Mar 25, 2004 |
|
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12153942 |
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Current U.S.
Class: |
514/1.1 ;
435/252.3; 435/320.1; 530/328; 536/23.1 |
Current CPC
Class: |
C07K 14/43518 20130101;
A61P 43/00 20180101; A61P 9/06 20180101; A61K 38/00 20130101; A61P
9/00 20180101 |
Class at
Publication: |
514/16 ; 530/328;
536/23.1; 435/320.1; 435/252.3 |
International
Class: |
A61K 38/08 20060101
A61K038/08; C07K 7/06 20060101 C07K007/06; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 1/21 20060101
C12N001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 2003 |
JP |
2003-085666 |
Claims
1. A purified polypeptide or salt thereof, the polypeptide
consisting of: an amino acid of SEQ ID NO: 2 an amino acid of SEQ
ID NO: 16 or an amino acid of SEQ ID NO: 17.
2. The polypeptide or salts in accordance with claim 1, wherein the
polypeptide having an intermolecular disulfide bond between two of
the cysteines contained in the amino acid of SEQ ID NO: 2, the
amino acid of SEQ ID NO: 16 or the amino acid of SEQ ID NO: 17.
3. A purified polypeptide or salts thereof, the polypeptide
consisting of: an amino acid of SEQ ID NO: 2 of which one or two
amino acids have been deleted, one or two amino acids have been
substituted, one or two amino acids have been inserted or one or
two amino acids have been added; the polypeptide having an
intermolecular disulfide bond between two of the cysteines; and the
polypeptide having mechano-sensitive channel inhibiting
activity.
4. A mechano-sensitive channel inhibitor comprising the purified
polypeptide or salt thereof as described in claim 1.
5. A mechano-sensitive channel inhibitor comprising the purified
polypeptide or salt thereof as described in claim 2.
6. A mechano-sensitive channel inhibitor comprising the purified
polypeptide or salt thereof as described in claim 3.
7. A remedy of atrial fibrillation comprising the step of
administering one of the purified polypeptide or salts thereof
described in claim 1.
8. A remedy of atrial fibrillation comprising the step of
administering one of the purified polypeptide or salts thereof
described in claim 2.
9. A remedy of atrial fibrillation comprising the step of
administering one of the purified polypeptide or salts thereof
described in claim 3.
10. A polynucleotide consisting of the polynucleotide that encodes
the polypeptide described in claim 1.
11. A polynucleotide consisting of the polynucleotide that encodes
the polypeptide described in claim 2.
12. A polynucleotide consisting of the polynucleotide that encodes
the polypeptide described in claim 3.
13. A recombinant vector comprising the polynucleotide described in
claim 10.
14. A recombinant vector comprising the polynucleotide described in
claim 11.
15. A recombinant vector comprising the polynucleotide described in
claim 12.
16. A transformant transformed with the recombinant vector
described in claim 10.
17. A transformant transformed with the recombinant vector
described in claim 11.
18. A transformant transformed with the recombinant vector
described in claim 12.
Description
[0001] This application is a divisional application of U.S. Ser.
No. 10/550,102, filed Sep. 21, 2005, which is a national stage of
International Application No. PCT/JP2004/004190 filed on Mar. 25,
2004, which is hereby incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a polypeptide that inhibits
the activity of a mechano-sensitive channel, and relates to a
mechano-sensitive channel inhibitor and remedy for atrial
fibrillation comprising the polypeptide. More specifically, the
present invention determines the pharmacophore that acts upon the
mechano-sensitive channel based on the sequence of a natural
peptide from spider venom (GsMTx-4), and it relates to novel
polypeptides designed to compose the pharmacophore which are
moreover useful for atrial fibrillation treatment.
BACKGROUND OF ART
[0003] Atrial-fibrillation is-a type of arrhythmia, of which the
morbidity prevalence rate increases with advanced age. Atrial
fibrillation is a heart disease observed in 3% of the elderly (over
65 years old). When atrial fibrillation becomes chronic, it forms a
thrombosis and induces cerebral thrombosis, and therefore it is
currently thought that atrial fibrillation is the main disease
factor of serious cases of cerebral apoplexy. Thus, considering the
frequency and seriousness of complications such as cerebral
infarction, atrial fibrillation has come to be regarded in recent
years as one kind of lethal arrhythmia (J. Nippon. Med. Sch. 2002,
69(3)). Heretofore, a remedy that completely cures atrial
fibrillation had not been obtained, and so it had been assumed that
medication for atrial fibrillation, particularly chronic atrial
fibrillation, had its limits (J. Nippon. Med. Sch. 2002,
69(3)).
[0004] Atrial fibrillation is believed to be caused in part by the
malfunctioning of an ion channel in the myocardium. Meanwhile, a
natural peptide from spider venom (GsMTx-4: SEQ ID NO:4) is known
to inhibit the activity of a mechano-sensitive channel
(Stretch-Activated Channel: SAC) (see for example Thomas M. Suchyna
et al., Identification of a Peptide Toxin from Grammostola
Spatulata Spider Venom that Blocks Cation-selective
Stretch-activated Channels, J. Gen. Physiol., Vol. 115, pp583-598
(2000) (non-patent document 1)). In the said document, it is
described that in peptides composing toxins derived from venoms
from terrestrial and aquatic animals, an ICK (Inhibitor Cysteine
Knot) motif with six cysteines is commonly observed (non-patent
document 1, p.590, right column, 11.7 to 3 from bottom, and FIG.
30). The said document also suggests that GsMTx-4 has an ICK motif
with a basic structure defined by three cysteine pairs
(C.sub.1-C.sub.4, C.sub.2-C.sub.5 and C.sub.3-C.sub.6) (non-patent
document 1, p.595, left column, 1.7 to 1.11 from bottom, column
`The structure of GsMTx-4`).
[0005] Furthermore, methods for extracting and purifying GsMTx-4,
methods for treating cerebral arrhythmia with the said GsMTx-4 and
so forth have been suggested (see for example Bode et al., Nature,
Vol.409, pp35-36 (2001) (non-patent document 2), US Patent
Application Published Description No.2002/0077286 (patent document
1)). Further, the structure of GsMTx-4 has been known from results
obtained in a solution using NMR (see Robert et al., J. Biol. Chem.
Vol.37, pp3443-3445, 2002. (non-patent document 3)). Despite such
findings, a remedy for atrial fibrillation using the peptide
derived from spider venom (GsMTx-4) had not been developed.
REFERENCES
[0006] Patent document 1: US Patent Application Published
Description No.2002/0077286;
[0007] Non-patent document 1: Thomas M. Suchyna et.al.,
Identification of a Peptide Toxin from Grammostola Spatulata Spider
Venom that Blocks Cation-selective Stretch-activated Channels, J.
Gen. Physiol., Vol.115, pp583-598 (2000);
[0008] Non-patent document 2: Bode et al., Nature, Vol.409, pp35-36
(2001);
[0009] Non-patent document 3: Robert et al., J. Biol. Chem. Vol.37,
pp3443-3445 (2002).
[0010] The object of the present invention is to identify the
pharmacophore (the minimum space structure needed for activation)
of GsMTx-4, to design novel polypeptides that specifically inhibit
the activity of a mechano-sensitive channel based on the
pharmacophore information, and to provide remedies for atrial
fibrillation comprising such polypeptides.
SUMMARY OF INVENTION
[0011] The above objects are achieved by the following
inventions.
[1] In a first aspect of the present invention, it involves a
polypeptide or salts thereof` consisting of an amino acid sequence
represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. These
polypeptides, as confirmed in the embodiment of this description,
are polypeptides that show mechano-sensitive channel inhibiting
activity, and can be considered as polypeptides that compose the
pharmacophore of GsMTx-4. These polypeptides are useful for
treatment of atrial fibrillation and such. [2] In a second aspect
of the present invention, it involves a polypeptide or salts
thereof comprising an amino acid sequence represented by SEQ ID
NO:1, SEQ ID NO:2 or SEQ ID NO:3. [3] In a third aspect of the
present invention, it involves a polypeptide or salts thereof
consisting of an amino acid sequence represented by SEQ ID NO:16 or
SEQ ID NO:17. These polypeptides, as confirmed in the embodiment of
this description, are polypeptides that show mechano-sensitive
channel inhibiting activity. These polypeptides are useful for
treatment of atrial fibrillation and such. [4] In a fourth aspect
of the present invention, it involves a polypeptide or salts
thereof consisting of an amino acid sequence represented by SEQ ID
NO:1, SEQ ID NO: 2 or SEQ ID NO:3 of which one or more of the amino
acids thereof have been deleted, substituted, inserted or added,
but not comprising an amino acid sequence described in SEQ ID NO:4,
and moreover showing mechano-sensitive channel inhibiting activity.
[5] In a fifth aspect of the present invention, it involves a
polypeptide or salts thereof described in the above [4] as
consisting of an amino acid sequence represented by SEQ ID NO:1,
SEQ ID NO:2 or SEQ ID NO:3 of which one or more of the amino acids
thereof have been deleted, substituted, inserted or added, but not
comprising an amino acid sequence described in SEQ ID NO:4, and
moreover showing mechano-sensitive channel inhibiting activity, of
which the said polypeptide comprises an amino acid sequence
represented by SEQ ID NO:16 or SEQ ID NO:17. [6] In a sixth aspect
of the present invention, it involves a polynucleotide comprising a
polynucleotide that encodes the polypeptide described in the above
[1], the above [3] or the above [4]. [7] In a seventh aspect of the
present invention, it involves a recombinant vector comprising the
polynucleotide described in the above [6]. [8] In a eighth aspect
of the present invention, it involves a transformant transformed by
the recombinant vector described in the above [7]. [9]0 In a ninth
aspect of the present invention, it involves a mechano-sensitive
channel inhibitor comprising one or more of the polypeptides or
salts thereof described in one of the above [1] to [5]. This
inhibitor specifically inhibits the activity of a mechano-sensitive
channel and thus is useful for conducting researches on
mechano-sensitive channels and such. [10] In a tenth aspect of the
present invention, it involves a remedy for atrial fibrillation
comprising one or more of the polypeptides or salts thereof
described in one of the above [1] to [5]. These polypeptides, as
confirmed of their functions in the embodiment of this description,
show mechano-sensitive channel inhibiting activity. Therefore, this
remedy can be used effectively in treating atrial fibrillation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a representation of the ten prospective multiple
alignments showing high homology to GsMTx-4
[0013] FIG. 2 is a representation of the alignment of 1QK6 and
GsMTx-4.
[0014] FIG. 3 is a stereo view superimposing Huwentoxin-I and
GsMTx-4.
[0015] FIG. 4 is a C.alpha. trace of the model of the superimposed
Huwentoxin-I and GsMTx-4. In FIG. 4, the thin lines indicate the
template and the thick lines indicate GsMTx-4.
[0016] FIG. 5 is a representation of the vicinity of Arg20
considered to be the activity center of Huwentoxin-I. In FIG. 5,
the thin lines indicate Huwentoxin-I and the thick lines indicate
GsMTx-4.
[0017] FIGS. 6(a) to 6(d) are representations of the surface
structure of a model with Huwentoxin-I (PDB code: 1QK6) as the
template. In FIGS. 6(a) to 6(d), the left side indicates
Huwentoxin-I, and the right side indicates GsMTx-4. FIG. 6(a) is a
view of the model from a hydrophobic patch (approximately the same
direction as the drawings above). FIG. 6(b) is a view of the model
rotated +90.degree. around the x-axis. FIG. 6(c) is a view of the
model rotated +90.degree. around the y-axis. FIG. 6(d) is a view of
the model rotated 180.degree. around the y-axis.
[0018] FIG. 7 is a representation of the structure of GsMTx-4 and
the structures of the designed peptides.
[0019] FIGS. 8(a) and 8(b) are representations of the inhibiting
activity of TVP003. FIG. 8(a) is a representation of the single
channel current recordings. FIG. 8(b) represents the channel's open
probability (Po).
[0020] FIGS. 9(a) and 9(b) are representations of the inhibiting
activity of TVP004. FIG. 9(a) is a representation of the single
channel current recordings. FIG. 9(b) represents the channel's open
probability (Po).
[0021] FIGS. 10(a) and 10(b) are representations of the inhibiting
activity of TVP005. FIG. 10(a) is a representation of the single
channel current recordings. FIG. 10(b) represents the channel's
open probability (Po).
[0022] FIGS. 11(a) and 11(b) are representations of the inhibiting
activity of TVP017. FIG. 11(a) is a representation of the single
channel current recordings. FIG. 11(b) represents the channel's
open probability (Po).
[0023] FIGS. 12(a) and 12(b) are representations of the inhibition
activity of TVP019. FIG. 12(a) is a representation of the single
channel current recordings. FIG. 12(b) represents the channel's
open probability (Po).
[0024] FIGS. 13(a) to 13(c) are results of the study into the
specificity of the active peptide to a mechano-sensitive channel.
FIG. 13(a) is a representation of the single channel current
recordings related to the myocardial SA channel of TVP003. FIG.
13(b) is a representation of the single channel current recordings
related to the STREX-deletion-mutant of TVP0003. FIG. 13(c)
represents the channel's open probability (Po).
BEST MODE FOR CARRYING OUT THE INVENTION
(Polypeptides of the Present Invention)
[0025] The Polypeptides of the present invention consist of a
polypeptide consisting of an amino acid sequence represented by SEQ
ID NO:1, SEQ ID NO:2 or SEQ ID NO:3; a polypeptide comprising an
amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ
ID NO:3; a polypeptide consisting of an amino acid sequence
represented by SEQ ID NO:16 or SEQ ID NO:17; a polypeptide
consisting of an amino acid sequence represented by SEQ ID NO:1,
SEQ ID NO:2 or SEQ ID NO:3 of which one or more of the amino acids
have been deleted, substituted, inserted or added, but not
comprising an amino acid sequence described in SEQ ID NO:4, and
moreover showing mechano-sensitive channel inhibiting activity
(namely, the polypeptides involved in embodiments 1 to 5 of the
present invention).
[0026] Furthermore, the polypeptides of the present invention may
have a C-terminus in the form of a carboxyl group (--COOH), a
carboxylate (--COO.sup.-), an amide (--CONH.sub.2), an ester
(--COOR) or the like.
[0027] The polypeptides of the present invention include
polypeptides wherein the amino group at the methionine residue of
the N-terminus is protected with a protecting group. The
polypeptides of the present invention include polypeptides wherein
the N-terminal is cleaved in vivo and the G1n thus formed is
pyroglutaminated. The polypeptides of the present invention include
polypeptides wherein a substituent on a side chain is protected by
an appropriate protecting group. The polypeptides of the present
invention include synthetic polypeptides such as the so-called
glycoproteins having conjugated sugar chains.
[0028] The salts of the polypeptides of the present invention may
be salts in the form of physiologically acceptable salts with acids
or bases, preferably in the form of physiologically acceptable acid
addition salts. Examples of such salts include salts with inorganic
acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid,
sulfuric acid), and salts with organic acids (e.g. acetic acid,
formic acid, propionic acid, fumaric acid, maleic acid, succinic
acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic
acid, methanesulfonic acid, benzensulfonic acid).
(Composition of the Polypeptides of the Present Invention)
[0029] The polypeptides of the present invention may be prepared by
chemical synthesis, or may be manufactured using recombinant DNA
technology. To prepare the polypeptides of the present invention by
chemical synthesis, the publicly known methods may be used, for
example, the peptide of the present invention can be obtained by
methods using azide, acid chloride, acid anhydride, compound acid
anhydride, DCC, activated ester, Woodward's reagent K,
carbonylimidazole, deoxidization, DCC/HONB, BOP reagent (see for
example Bozanszky, M and M. A. Ondetti, Peptide Synthesis,
Interscience Publishers, New York (1966); Schroeder and Luebke, The
Peptide, Academic Press, New York (1 965); F. M. Finn and K.
Hofmann, The Proteins Vol.2, H. Nenrath, R. L. Hill ed., Academic
Press Inc., New York (1976); Nobuo Izumiya et al., Peptide Gosei no
Kiso to Jikken (Basics and experiments of peptide synthesis),
Maruzen Co. (1985); Haruaki Yajima and Shunpei Sakakibara et al.,
Seikagaku Jikken Koza (Biochemical Experiment) 1, Japanese
Biochemical Society ed., Tokyo Kagaku Dojin Co. (1977); Toshiya
Kimura, Zoku Seikagaku Jikken Koza (Sequel to Biochemical
Experiment) 2, Japanese Biochemical Society ed., Tokyo Kagaku Dojin
Co. (1987)). Furthermore, the peptide of the present invention can
be prepared by chemical synthesis using an automated peptide
synthesizer (e.g. PE Applied Bio Systems Co.).
[0030] Further, following the completion of reaction, the
polypeptides of the present invention can be purified and separated
by publicly known purification methods. For example, the
polypeptide of the present invention can be purified and separated
by a combination of solvent extraction, distillation, column
chromatography, liquid chromatography, recrystallization and the
like. Where the peptide of the present invention obtained by the
above methods is in a free form, publicly known methods can be used
to convert it into a salt form, and on the other hand, where the
peptide is obtained in a salt form, publicly known methods can be
used to convert it into a free form.
(Polynucleotide Encoding the Polypeptide)
[0031] The polynucleotide encoding the polypeptide of the present
invention may be any polynucleotide so long as it contains the base
sequence (DNA or RNA, preferably DNA) encoding the polypeptide of
the present invention. For example, the polynucleotide may be the
DNA or RNA such as mRNA encoding the polypeptide of the present
invention, and it can either be double stranded or single stranded.
When double stranded, it may be a double stranded DNA, a double
stranded RNA or a hybrid of DNA and RNA. When single stranded, it
may either be a sense strand (namely, a coding strand) or an
anti-sense strand (namely, a non-coding strand).
[0032] Using the polynucleotide encoding the polypeptide of the
present invention, mRNA of the polypeptide of the present invention
can be assayed, for example, according to the publicly known method
described in the special issue of Jikken Igaku (Experimental
Medicine), Shin PCR to Sono Oyo (Novel PCR and. its application)
15(7), 1997, or according to a similar method.
[0033] The DNA encoding the polypeptide of the present invention
include genomic DNA, genomic DNA library, cDNA derived from the
cells or tissues described above, cDNA library derived from the
cells and tissues described above, and synthetic DNA, of which any
one thereof can be employed. Examples of the vector used for the
library include bacteriophage, plasmid, cosmid and phagemide, of
which any one thereof can be employed. Further, the DNA can be
directly amplified by Reverse Transcriptase Polymerase Chain
Reaction (hereinafter abbreviated as RT-PCR) employing a total RNA
or a mRNA fraction prepared from the cells or tissues described
above.
[0034] For cloning the DNA encoding the polypeptide of the present
invention, there is the method of amplifying by PCR using synthetic
DNA primers comprising a part of the base sequence of the DNA
encoding the polypeptide of the present invention. The cloning of
the DNA encoding the polypeptide of the present invention can also
be performed by selecting the DNA inserted into an appropriate
vector by hybridization with a labeled DNA fragment or synthetic
DNA that encodes a part of the region or the entire region of the
polypeptide of the present invention. Hybridization can be carried
out, for example, according to the method described in Molecular
Cloning 2nd (J. Sambrook et al., Cold Spring Harbor Lab. Press,
1989). When using commercially available library, hybridization can
be carried out according to the method described in the attached
instructions.
[0035] Substitution of the base sequence of the DNA can be carried
out by PCR or by using publicly known kits, for example,
Mutan.TM.-super Express Km (Takara Shuzo Co.) and Mutan.TM.-K
(Takara Shuzo Co.) according to publicly known methods such as
ODA-LA PCR, Gapped duplex method and Kunkel method, or according to
similar methods.
[0036] The cloned DNA encoding the polypeptides can be used as it
is, or if desired, used after digesting with a restriction enzyme
or after adding a linker thereto. Such DNA can contain ATG as
translation initiation codon at the 5' end thereof and can contain
TAA, TGA or TAG as translation termination codon at the 3' end
thereof. These translation initiation codon and translation
termination codon can be added by using an appropriate synthetic
DNA adapter.
[0037] The expression vector of the polypeptide of the present
invention can be manufactured, for example, by excising a desired
DNA fragment from the DNA encoding the polypeptide of the present
invention and then litigating the DNA fragment downstream of a
promoter in an appropriate expression vector.
[0038] Examples of the vector include plasmids derived from a
Escherichia coli (e.g., pCR4, pCR2.1, pBR322, pBR325, pUC12,
pUC13), plasmids derived from Bacillus subtlis (e.g., pUB110, pTP5,
pC194), plasmids derived from yeast (e.g., pSH19, pSH15),
bacteriophages such as .sub..lamda. phage, animal viruses such as
retrovirus, vaccinia virus and baculovirus, as well as pA1-11,
pXT1, pRc/CMV, pRc/RSV and pcDNAI/Neo.
[0039] The promoter employed in the present invention can be any
promoter so long as it is an appropriate promoter matching the host
used for gene expression. For example, when using animal cells as
the host, SR.alpha. promoter, SV40 promoter, LTR promoter, CMV
promoter, HSV-TK promoter or such can be utilized.
[0040] Of these, it is preferable to use CMV promoter, SR.alpha.
promoter or the like. When using bacteria of the genus Escherichia
as the host, the preferred promoter is trp promoter, 1ac promoter,
recA promoter, .sub..lamda.P.sub.L promoter, 1pp promoter or the
like, when using bacteria of the genus Bacillus as the host, the
preferred promoter is SPO1 promoter, SPO2 promoter, penP promoter
or the like, when using yeast as the host, the preferred promoter
is PHO5 promoter, PGK promoter, GAP promoter, ADH promoter or the
like. When using insect cells as the host, it is preferable to use
polyhedron promoter, P10 promoter or the like.
[0041] For the expression vector, in addition to the above
examples, those comprising an enhancer, a splicing signal, a poly A
addition signal, a selection marker, an SV40 replication origin
(hereafter may be abbreviated as SV40Ori) or such can be used if so
desired. Examples of the selection marker include dihydrofolate
reductase (hereinafter may be abbreviated as dhfr) gene
(methotrexate (MTX) resistance), amplicillon resistant gene
(hereinafter may be abbreviated as Amp.sup.r), neomycin resistant
gene (hereinafter may be abbreviated as Neo.sup.r, G418 resistance)
and the like. In particular, when using dhfr gene as the selection
marker by employing CHO (dhfr.sup.-) cells, the desired gene may be
selected using a medium not comprising thymidine.
[0042] When necessary, a signal sequence matching the host is added
to the N-terminus of the polypeptide of the present invention. When
using bacteria of the genus Escherichia as the host, PhoA signal
sequence, OmpA signal sequence or such can be utilized, when using
bacteria of the genus Bacillus as the host, .alpha.-amylase signal
sequence, subtilisin signal sequence or such can be utilized, when
using yeast as the host, MF.alpha. signal sequence, SUC2 signal
sequence or such can be utilized, and when using animal cells as
the host, insulin signal sequence, .alpha.-interferon signal
sequence, antibody molecule signal sequence or such can be
utilized.
[0043] Using the vector comprising the DNA encoding the polypeptide
of the present invention thus composed, transformants can be
manufactured.
[0044] Examples of the host include bacteria of the genus
Escherichia, bacteria of the genus Bacillus, yeast, insect cells,
insect and animal cells.
[0045] Specific examples of the bacteria of the genus Escherichia
include Escherichia coli K12DH1 (Proc. Natl. Acad. Sci. USA, 60,
160 (1968)), JM103 (Nucleic Acids Research, 9, 309 (1981)), JA221
(Journal of Molecular Biology, 120, 517 (1978)), HB101 (Journal of
Molecular Biology, 41, 459 (1969)), C600 (Genetics, 39, 440
(1954)), DH5.alpha. (Inoue, H., Nojima, H. and Okayama, H., Gene,
96, 23-28 (1990)), and DH10B (Proc. Natl. Acad. Sci. USA, 87,
4645-4649 (1990)).
[0046] Examples of the bacteria of the genus Bacillus include
Bacillus subtilis Mi114 (Gene, 24, 255 (1983)), and 207-21 (Journal
of Biochemistry, 95, 87 (1984)).
[0047] Examples of yeast include Saccharomyces cerevisiae AH22,
AH22R--, NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe
NCYC1913, NCYC2036, and Pichia pastoris.
[0048] For insect cells, where the virus is AcNPV, Spodoptera
frugiperda cells (Sf cells), MG1 cells derived from the
mid-intestine of Trichoplusia ni, HighFive.TM. cells derived from
the egg of Trichoplusoa ni, cells derived from Mamestra brassicae,
cells derived from Estigmena acrea and the like can be used. Where
the virus is BmNPV, Bombyx mori N cells (BmN cells) and the like
can be used. Examples of the Sf cells thereof include Sf9 cells
(ATCC CRL1711), and Sf21 cells (see Vaughn, J. L. et al., In Vivo,
13, 213-217 (1977) for above).
[0049] For insects, the larva of silkworms can be used (Maeda et
al., Nature, 315, 592 (1985)).
[0050] Examples of animal cells include monkey cells COS-7, Vero,
Chinese hamster cells CHO (hereinafter abbreviated as CHO cells),
dhfr gene deficient Chinese hamster cells CHO (hereinafter
abbreviated as CHO(dhfr.sup.-) cells), mouse L cells, mouse AtT-20,
mouse myeloma cells, rat GH3, and human FL cells.
[0051] Bacteria of the genus Escherichia can be transformed
according to the method described, for example, in Proc. Natl.
Acad. Sci. USA, 69, 2110 (1972) or in Gene, 17 107 (1982).
[0052] Bacteria of the genus Bacillus can be transformed according
to the method described, for example, in Molecular & General
Genetics, 168, 111 (1979).
[0053] Yeast can be transformed according to the method described,
for example, in Methods in Enzymology, 194, 182-187 (1991) or in
Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)
[0054] Insect cells and insects can be transformed according to the
method described, for example, in Bio/Technology, 6, 47-55
(1988)
[0055] Animal cells can be transformed according to the method
described, for example, in Saibo Kogaku (Cell Engineering) extra
issue 8, Shin Saibo Kogaku Jikken Protocol (New Cell Engineering
Experimental Protocol), 263-267, Shujunsha (1995) or in Virology,
52, 456 (1973).
[0056] Thus, a transformant transformed with the expression vector
comprising the DNA encoding the polypeptide of the present
invention can be obtained.
[0057] In cultivating the transformant having bacteria of the genus
Escherichia or bacteria of the genus Bacillus as the host, the
appropriate medium used for cultivation is a liquid medium wherein
carbon sources, nitrogen sources, inorganic substances and such
required for the growth of the said transformant are contained.
Carbon sources include glucose, dextrin, soluble starch, sucrose
and the like, nitrogen sources include inorganic or organic matter
such as ammonium salts, nitrate salts, corn steep liquor, peptone,
casein, meat extract, soybean cake, potato extract and the like,
and inorganic substances include calcium chloride, sodium
dihydrogenphosphate, magnesium chloride and the like. In addition,
yeast extract, vitamins, growth promoting factors and such can be
added. The pH of the medium is preferably about 5 to 8.
[0058] The preferred medium for cultivating bacteria of the genus
Escherichia is M9 medium comprising glucose and Casamino acids
(Miller, Journal of Experiments in Molecular Genetics, 431-433,
Cold Spring Harbor Laboratory, New York (1972)) or the like. When
necessary, chemicals such as 3.beta.-indolylacrylic acids can be
added therein to make the promoter function efficiently.
[0059] When using bacteria of the genus Escherichia as the host,
cultivation is generally carried out at about 15 to 43.degree. C.
for about 3 to 24 hours, and aeration or agitation can be conducted
when necessary.
[0060] When using bacteria of the genus Bacillus as the host,
cultivation is generally carried out at about 30 to 40.degree. C.
for about 6 to 24 hours, and aeration or agitation can be conducted
when necessary.
[0061] When cultivating a transformant with yeast as the host, the
medium to be used is, for example, Burkholder's minimal medium
(Bostian, K. L. et al., Proc. Natl. Acad. Sci. USA, 77, 4505
(1980)), or SD medium comprising 0.5% Casamino acids (Bitter, G A.
et al., Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)). Preferably,
pH of the medium is adjusted to about 5 to 8. Cultivation is
generally carried out at about 20 to 35.degree. C. for about 24 to
72 hours, and aeration or agitation is conducted when
necessary.
[0062] When cultivating a transformant with insect cells or insects
as the host, the medium to be used is, for example, Grace's Insect
Medium (Grace, T. C. C., Nature, 195,788 (1962)) to which is added
appropriate amounts of additives such as immobilized 10% calf
serum. Preferably, pH of the medium is adjusted to about 6.2 to
6.4. Cultivation is generally carried out at about 27.degree. C.
for about 3 to 5 days, and aeration or agitation is conducted when
necessary.
[0063] When cultivating a transformant with animal cells as the
host, the medium to be used is, for example, MEM medium comprising
about 5 to 20% of fetal calf serum (Science, 122, 501 (1952)), DMEM
medium (Virology, 8, 396 (1959)), RPMI 1640 medium (Journal of the
American Medical Association, 199, 519 (1967)), or 199 medium
(Proceeding of the Society for the Biological Medicine, 73, 1
(1950)) Preferably, pH should be about 6 to 8. Cultivation is
carried out at about 30 to 40.degree. C. for about 15 to 60 hours,
and aeration or agitation is conducted when necessary.
[0064] In the foregoing manner, the polypeptide of the present
invention can be produced in the transformant intracellularly, in
cell membranes, or extracellularly.
[0065] The polypeptide of the present invention can be separated
and purified from the above culture medium using the methods
described below.
[0066] When extracting the polypeptide of the present invention
from the cultured bacteria or cells, appropriate methods are used
in which following cultivation, bacteria or cells are collected by
publicly known methods and suspended in an appropriate buffer, the
bacteria or cells are then disrupted by ultrasound, lysozyme and/or
freeze-thawing, after which the crude extract of the polypeptide is
obtained through centrifugation or filtration. The buffer may
contain a protein modifier such as urea or guanidine hydrochloride,
or a surfactant such as Triton X-100.TM.. Where the polypeptide is
secreted into the culture broth, following the completion of
cultivation, the bacteria or cells are separated from the
supernatant using a publicly known method, and the supernatant is
collected.
[0067] The polypeptide contained in the cultured supernatant or the
extract thus obtained can be purified by appropriately combining
publicly known separation and purification methods. Such publicly
known separation and purification methods include methods that make
use of solubility such as salting out and solvent precipitation,
methods that make use of difference in molecular weight such as
dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide
gel electrophoresis, methods that make use of difference in
electric charge such as ion exchange chromatography; methods that
make use of the difference in specific affinity such as affinity
chromatography, methods that make use of difference in
hydrophobicity such as reverse phase high performance liquid
chromatography, and methods that make use of difference in
isoelectric point such as isoelectrofusing electrophoresis.
[0068] Where the polypeptide thus obtained is in a free form,
publicly known methods or similar methods can be used to convert it
into a salt form, and on the other hand, where it is obtained in a
salt form, publicly known methods or similar methods can be used to
convert it into a free form or into another salt form.
[0069] Further, the polypeptide produced by the recombinant can be
optionally modified and parts of the polypeptide can be removed by
activating an appropriate protein modifying enzyme before or after
purification. Examples of the protein-modifying enzyme include
trypsin, chymotrypsin, arginyl endopeptidase, protein kinase and
glycosidase.
[0070] The activity of the polypeptide of the present invention or
salts thereof thus generated can be measured by a binding
experiment to a labeled ligand, an enzyme immunoassay using a
specific antibody, or such.
(Mechano-Sensitive Channel Inhibitors)
[0071] A mechano-sensitive channel inhibitor contains, for example,
one or more of either the polypeptides of the present invention or
salts thereof (hereinafter may be described as polypeptides of the
present invention). The polypeptides of the present invention can
be utilized as mechano-sensitive channel inhibitors. The
polypeptides of the present invention are easy to handle, and as
shown in the embodiments below, display high inhibiting
activity.
(Remedies for Atrial Fibrillation)
[0072] A remedy for atrial fibrillation contains, for example, one
or more of either the polypeptides of the present invention or
salts thereof. In other words, the present invention can provide
pharmaceuticals and pharmaceutical compositions as well.
Pharmaceutical compositions are, for example, those that contain
the polypeptide of the present invention or salts thereof and a
pharmaceutically acceptable carrier.
[0073] The remedy for atrial fibrillation comprising the
polypeptide of the present invention can be administered
parenterally, for example, into the blood vessel of the heart in
the form of an injection, or can be used orally, for example, in
the form of tablets or capsules. Formulations for injection can be
provided in ampules comprising a unit dosage or in containers
comprising multiple dosages. Moreover, the preparations can be
administered not only to humans but also to other warm-blooded
animals. The formulations can be prepared using publicly known
preparation methods.
[0074] The various preparations can be manufactured using
conventional methods by appropriately selecting generally used
formulations such as an excipient, a swelling agent, a binder, a
moistening agent, a disrupting agent, a lubricant, a surface-active
agent, a dispersing agent, a buffer, a preservative, a solubilizing
agent, an antiseptic, a sweetening and flavoring agent, a soothing
agent, a stabilizing agent, and an isotonic agent. The various
agents described above may also contain pharmaceutically acceptable
carriers or additives. Such carriers or additives include water,
pharmaceutically acceptable organic solvents, collagen, polyvinyl
alcohol, polyvinyl pyrrolidone, carboxyvinylpolymer, alginic acid
sodium, water-soluble dextran, carboxymethyl starch sodium, pectin,
Xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene
glycol, polyethylene glycol, Vaseline, paraffin, stearate alcohol,
stearic acid, human serum albumin, mannitol, sorbitol, and lactose.
The additives to be used are selected appropriately or in
combination from those described above according to the
formulations of the present invention.
[0075] The polypeptide of the present invention consisting of the
activated component in the forms described above is contained
therein, for example, 0.01 to 100% by weight, preferably 0.1 to 90%
by weight, more preferably 1 to 50% by weight.
[0076] Concerning the dosage of the polypeptide of the present
invention, when administered parenterally, the amount of one dosage
differs depending on the subject of administration, symptoms and
route of administration; and so, if administered in the form of
injection to a patient (weighing 60 kg) with atrial fibrillation,
for example, the daily dosage is generally about 0.01 to 30 mg,
preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg.
When administered orally to an atrial fibrillation patient
(weighing 60 kg), for example, the daily dosage is about 0.1 to 100
mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20
mg. The remedy for atrial fibrillation of the present invention is
preferably administered once to several times a day for a duration
of more than one day.
(Identification of the Pharmacophore)
[0077] In designing the polypeptide of the present invention, the
region of the peptide from spider venom (GsMTx-4) consisting of the
pharmacophore that is the minimal unit necessary for activation has
been estimated with good precision based on a three-dimensional
structure.
[0078] The pharmacophore can be identified for example in the
manner described hereinafter. First, a precise structure estimation
of the peptide from spider venom is conducted according to the
homology modeling method using a similar peptide with a known
three-dimensional structure as the template. Based on the structure
thus obtained, a function analysis of the peptide with a modified
activated part is performed and the target part for pharmaceutical
design is narrowed down. A design that mimics the disulfide bond
region of GsMTx-4 is created, and the peptide with a stable
structure is designed. GsMTx-4 is known to have a three-dimensional
structure of relatively low flexibility that comprises three
disulfide bonds, and so the pharmacophore needed for activation is
identified by designing several cyclic peptides comprising polar
amino acid residues generally often involved in the binding
process.
(Measurement of the Activity)
[0079] The activity evaluation method of the peptide of the present
invention can be carried out using the publicly known activity
evaluation method, preferably using the single channel current
recording method employing the patch clamp method described in
embodiment 1.
Embodiment 1
Example 1: Search for a Similar Peptide with a Known Structure
Showing High Homology to the Sequence of the Peptide from the
Spider Venom (GsMTx-4)
[0080] A structure with high homology to the amino acid sequence of
GsMTx-4 represented by SEQ ID NO:4 was searched among the spider
venoms from PDB (Protein Data Bank). Consequently, 10 candidates
with high homology to GsMTx-4 were found. The results of the
multiple alignment of those candidates are shown in FIG. 1
below.
[0081] Of the sequences shown in FIG. 1, 1QK6 (Huwentoxin-I: SEQ ID
NO:12) comprising the same cysteine residues, and moreover having
almost the same length between the cysteine residues with no
insertion or deletion (GsMTx-4 is one residue longer) was selected
as the template to narrow down the pharmacophore using the homology
modeling method.
Example 2: Estimation of the Three Dimensional Structure of the
Peptide of Spider Venom (GsMTx-4).
[0082] Homology modeling was carried out using the template peptide
1QK6. First, alignment of 1QK6 and GsMTx-4 was performed. The
result is shown in FIG. 2. Next, the model structure was
constructed with program MODELLER. The superimposition of the
constructed structure model of GsMTx-4 and Huwentoxin-I used as the
template is shown in FIG. 3 and in FIG. 4. FIG. 3 is a stereo view
of the superimposed Huwentoxin-I and GsMTx-4. FIG. 4 is a C.alpha.
trace of the model of the superimposed Huwentoxin-I and GsMTx-4. In
FIG. 4, the thin lines indicate the template, and the thick lines
indicate GxMTx-4.
[0083] The vicinity of Arg20 considered to be the activity center
of Huwentoxin-I is shown in FIG. 5. In FIG. 5, the thin lines
indicate Huwentoxin-I, and the thick lines indicate GsMTx-4.
Furthermore, comparisons of the surface structures of Huwentoxin-I
and GsMTx-4 are shown in FIG. 6. In FIGS. 6(a) to 6(d, the left
side shows Huwentoxin-I, and the right side shows GsMTx-4. FIG.
6(a) is a view from a hydrophobic patch (approximately the same
direction as the drawings above). FIG. 6(b) is a view rotated
+90.degree. around the x-axis. FIG. 6(c) is a view rotated
+90.degree. around the y-axis. FIG. 6(d) is a view rotated
180.degree. around the y-axis. FIG. 6(b) shows that the molecules
of the two peptides have quite different forms. The distributions
of the residues with isolable side chains also differ, and can be
assumed to be concerned with the determination of specificity.
[0084] Further, by comparing the structure of GsMTx-4 obtained in a
solution using NMR disclosed in Robert et. al., J. Biol. Chem.
Vol.37, pp3443-3445, 2002 (non-patent document 3 mentioned above)
and the structure of GsMTx-4 obtained in the present embodiment,
the modeling structure constructed in the present invention can be
judged as reflecting the actual structure of GsMTx-4.
Example 3: Design of the Active Peptide and Identification of the
Pharmacophore Thereof
[0085] Based on the modeling structure of GsMTx-4 described above,
the policy for designing the peptide fragments was decided. The
structure of GsMTx-4 and the designed structure of the peptides are
shown in FIG. 7. GsMTx-4 comprises four loops formed by three
disulfide bonds as indicated in the sequence structure formula
shown in FIG. 7. Therefore, five peptides TVP001 to TVP005 were
designed to determine the loop contributing to the inhibiting
activity. In these peptides, cysteines at the regions not composing
the loops have been substituted with alanines. TVP011 (SEQ ID
NO:14) comprises loops 1 and 2, TVP002 (SEQ ID NO:15) comprises
loops 3 and 4, TVP003 (SEQ ID NO:1) comprises loop 2, TVP004 (SEQ
ID NO:2) comprises loop 3, and TVP005 (SEQ ID NO:3) comprises loops
2 and 3.
(Bioassay of the Designed Peptides)
[0086] Activity assay of the peptides was carried out by the most
reliable single channel current recording method using the patch
clamp method. As the subject of the assay, Ca.sup.2+ dependant BigK
channel (Kawakubo et. Am J Physiol, 276 : H1827.1999) derived from
heart muscle was employed. By applying this channel to the
expressed ventricle muscle of chicken or to CHO cells
force-expressing the cDNA of this channel with the cell-attached
patch clamp method, an inside-out excised patch was formed, and
single channel current recordings were taken under a fixed membrane
potential. Assuming that the designed synthetic peptide blocks the
channel extracellularly the same as GsMTx-4, the space a fixed
distance above the glass pipette used for recording was filled with
the peptide of a known concentration beforehand, and administration
was carried out by back-fill which utilizes diffusion to reach the
channel, With this method, the concentration of the peptide inside
the pipette reaches equilibrium 15 to 20 minutes after the start of
diffusion, and so the dissociation constant of the peptide can be
estimated from the inhibition ratio at 20 minutes and onwards. Or,
the relative inhibition of the peptide can be estimated from the
time taken for inhibition. If the exact dissociation constant of
the peptide is to be calculated, it is necessary to prepare an
outside-out excised patch, take single channel current recordings,
analyze the inhibition effects of various peptides with different
concentration, and obtain a dose-inhibition curve; however, as this
method requires special technology and as this is the primary
screening of the various designed synthetic peptides, the assay
method combining the inside-out excised patch and back-fill
described above was used to make a rough estimate of the inhibition
effects of the peptides. Taking into consideration the results
obtained for GsMTx-4, the concentration of the peptide was
maintained at 10 .mu.M. Concerning the scale of evaluation, the
channel's open probability (Po, indicated in percentage) was used,
and the level of inhibition was expressed by the inhibition ratio
(percentage) with the control (prior to the administration of the
peptide) as the standard, or by the time taken for inhibition.
(Results of the Assay)
[0087] Of the five designed synthetic peptides, TVP003, TVP004 and
TVP005 displayed inhibiting activity. Therefore, mutation TVP017
(SEQ ID NO:16) in which the 3.sup.rd R from the N-terminus of
TVP004 had been substituted by A and mutation TVP019 in which the
7.sup.th K from the N-terminus of TVP004 had been substituted by A
were synthesized, and the inhibiting activity was measured using
the bioassay described above; as a result, these synthetic peptides
were also found to show inhibiting activity.
[0088] The inhibiting activity of TVP003 is shown in FIG. 8. FIG.
8(a) is a representation of single channel current recordings. FIG.
8(b) represents the channel's open probability (Po). As indicated
in FIG. 8, TVP003 showed 100% inhibition after 8 minutes, and so
the dissociation constant of TVP003 was estimated as .mu.M order or
lower. It is suggested from this value that TVP003 shows inhibiting
activity of a level the same as or higher than the natural peptide
from spider venom, GsMTx-4.
[0089] The inhibiting activity of TVP004 is shown in FIG. 9. FIG.
9(a) is a representation of the single channel recordings. FIG.
9(b) represents the channel's open probability (Po) As indicated in
FIG. 9, at 10 .mu.M, TVP004 showed inhibition activity of about 60%
after 8 minutes, and 95% after 16 minutes, displaying a relatively
high level of inhibition activity.
[0090] The inhibition activity of TVP005 is shown in FIG. 10. FIG.
10(a) is a representation-of the single channel recordings. FIG.
10(b) represents the channel's open probability (Po). As indicated
in FIG. 10, TVP005 showed about 60% inhibition after 20 minutes,
and so the dissociation constant thereof was estimated as
approximately about 10 .mu.M.
[0091] The inhibition activity of TVP017 is shown in FIG. 11. FIG.
11(a) is a representation of the single channel recordings. FIG.
11(b) represents the channel's open probability (Po). As indicated
in FIG. 11, TVP017 showed 100% inhibition after 6 minutes, and so
was found to display an even higher level of inhibition than
TVP003. It is suggested from this value that TVP017 shows
inhibition activity of a higher level than the natural peptide from
spider venom, GsMTx-4.
[0092] The inhibition activity of TVP019 is shown in FIG. 12. FIG.
12(a) is a representation of the single channel recordings. FIG.
12(b) represents the channel's open probability (Po). As indicated
in FIG. 12, TVP019 showed approximately 80% inhibition after 14
minutes.
Embodiment 2: Specificity of the Peptide to the Channel
Example 4: Specificity of the Active Peptide to a Mechano-Sensitve
Channel
[0093] The specificity of the peptide of the present invention to a
mechano-sensitive channel was examined. TVP003 was used as the
active peptide, and myocardial SA channel, the same as that
utilized in example 3, was used as the channel. This channel has a
STREX sequence consisting of 59 amino acids at the C-terminus, and
the STREX-deletion-mutant deleted of that sequence loses almost all
extension activity, thus becoming a common Ca dependant bigK
channel (SAKCA: Tang, Naruse, Sokabe, J Membr Biol, 169:185-200,
2003). FIG. 13 is the result of study into the specificity of the
active peptide to a mechano-sensitive channel. FIG. 13(a) is a
representation of the single channel recordings related to the
myocardial SA channel of TVP003. FIG. 13(b) is a representation of
the single channel recordings related to the STREX-deletion-mutant
of TVP003. FIG. 13(c) represents the channel's open probability
(Po). It can be seen from FIG. 13 that GsMTx-4 and TVP003 inhibit
the wild-type SA channel but do not inhibit the
STREX-deletion-mutant hardly at all. Therefore, GsMTx-4 and TVP003
can be considered as specifically acting only on a channel with
extension activity.
INDUSTRIAL APPLICABILITY
[0094] According to the present invention, it is possible to obtain
a novel polypeptide that specifically inhibits the activity of a
mechano-sensitive channel.
[0095] Utilizing a polynucleotide encoding such a polypeptide of
the present invention, a recombinant vector comprising this
polynucleotide, and a transformant transformed by the recombinant
vector, the polypeptide of the present invention can be produced in
large quantities.
[0096] A mechano-sensitive channel inhibitor comprising the
polypeptide of the present invention or salts of the polypeptide of
the present invention is useful for manufacturing a reagent related
to a mechano-sensitive channel.
[0097] A remedy for atrial fibrillation comprising the polypeptide
of the present invention or salts of the polypeptide of the present
invention can efficiently treat atrial fibrillation.
Sequence CWU 1
1
17110PRTArtificial SequenceDescription of Artificial
SequenceSynthetic Polypeptide 1Trp Lys Cys Asn Pro Asn Asp Asp Lys
Cys 1 5 1028PRTArtificial SequenceDescription of Artificial
SequenceSynthetic Polypeptide 2Cys Ala Arg Pro Lys Leu Lys Cys 1
5317PRTArtificial SequenceDescription of Artificial
SequenceSynthetic Polypeptide 3Trp Lys Cys Asn Pro Asn Asp Asp Lys
Ala Ala Arg Pro Lys Leu Lys 1 5 10 15Cys435PRTGrammostola spatulata
4Gly Cys Leu Glu Phe Trp Trp Lys Cys Asn Pro Asn Asp Asp Lys Cys 1
5 10 15Cys Arg Pro Lys Leu Lys Cys Ser Lys Leu Phe Lys Leu Cys Asn
Phe 20 25 30Ser Ser Gly 35542PRTAtrax robustus 5Cys Ala Lys Lys Arg
Asn Trp Cys Gly Lys Asn Glu Asp Cys Cys Cys 1 5 10 15Pro Met Lys
Cys Ile Tyr Ala Trp Tyr Asn Gln Gln Gly Ser Cys Gln 20 25 30Thr Thr
Ile Thr Gly Leu Phe Lys Lys Cys 35 40642PRTHadronyche versuta 6Cys
Ala Lys Lys Arg Asn Trp Cys Gly Lys Thr Glu Asp Cys Cys Cys 1 5 10
15Pro Met Lys Cys Val Tyr Ala Trp Tyr Asn Glu Gln Gly Ser Cys Gln
20 25 30Ser Thr Ile Ser Ala Leu Trp Lys Lys Cys 35
40730PRTHeteropodidae veratoria 7Asp Asp Cys Gly Lys Leu Phe Ser
Gly Cys Asp Thr Asn Ala Asp Cys 1 5 10 15Cys Glu Gly Tyr Val Cys
Arg Leu Trp Cys Lys Leu Asp Trp 20 25 30832PRTSelenocosmia huwena
8Gly Cys Leu Gly Asp Lys Cys Asp Tyr Asn Asn Gly Cys Cys Ser Gly 1
5 10 15Tyr Val Cys Ser Arg Thr Trp Lys Trp Cys Val Leu Ala Gly Pro
Trp 20 25 30937PRTAgelenopsis aperta 9Ala Cys Val Gly Glu Asn Gln
Gln Cys Ala Asp Trp Ala Gly Pro His 1 5 10 15Cys Cys Asp Gly Tyr
Tyr Cys Thr Cys Arg Tyr Phe Pro Lys Cys Ile 20 25 30Cys Arg Asn Asn
Asn 351037PRTAgelenopsis aperta 10Ala Cys Val Gly Glu Asn Gln Gln
Cys Ala Asp Trp Ala Gly Pro His 1 5 10 15Cys Cys Asp Gly Tyr Tyr
Cys Thr Cys Arg Tyr Phe Pro Lys Cys Ile 20 25 30Cys Arg Asn Asn Asn
351137PRTAgelenopsis apertaUNSURE(37)Xaa represents unknown amino
acid residue 11Glu Cys Val Pro Glu Asn Gly His Cys Arg Asp Trp Tyr
Asp Glu Cys 1 5 10 15Cys Glu Gly Phe Tyr Cys Ser Cys Arg Gln Pro
Pro Lys Cys Ile Cys 20 25 30Arg Asn Asn Asn Xaa
351233PRTSelenocosmia huwena 12Ala Cys Lys Gly Val Phe Asp Ala Cys
Thr Pro Gly Lys Asn Glu Cys 1 5 10 15Cys Pro Asn Arg Val Cys Ser
Asp Lys His Lys Trp Cys Lys Trp Lys 20 25 30Leu1337PRTSelenocosmia
huwena 13Leu Phe Glu Cys Ser Phe Ser Cys Glu Ile Glu Lys Glu Gly
Asp Lys 1 5 10 15Pro Cys Lys Lys Lys Lys Cys Lys Gly Gly Trp Lys
Cys Lys Phe Asn 20 25 30Met Cys Val Lys Val 351417PRTArtificial
SequenceDescription of Artificial SequenceSynthetic Polypeptide
14Gly Cys Leu Glu Phe Trp Trp Lys Ala Asn Pro Asn Asp Asp Lys Ala 1
5 10 15Cys1515PRTArtificial SequenceDescription of Artificial
SequenceSynthetic Polypeptide 15Cys Ala Arg Pro Lys Leu Lys Ala Ser
Lys Leu Phe Lys Leu Cys 1 5 10 15168PRTArtificial
SequenceDescription of Artificial SequenceSynthetic Polypeptide
16Cys Ala Ala Pro Lys Leu Lys Cys 1 5178PRTArtificial
SequenceDescription of Artificial SequenceSynthetic Polypeptide
17Cys Ala Arg Pro Lys Leu Ala Cys 1 5
* * * * *