U.S. patent application number 15/393315 was filed with the patent office on 2017-05-04 for coelenterazine analogs.
This patent application is currently assigned to JNC CORPORATION. The applicant listed for this patent is JNC CORPORATION. Invention is credited to Satoshi INOUYE, Yuiko MIURA, Junichi SATO.
Application Number | 20170121755 15/393315 |
Document ID | / |
Family ID | 50776858 |
Filed Date | 2017-05-04 |
United States Patent
Application |
20170121755 |
Kind Code |
A1 |
INOUYE; Satoshi ; et
al. |
May 4, 2017 |
COELENTERAZINE ANALOGS
Abstract
Coelenterazine analogs having luminescence properties different
from those of known coelenterazine analogs are desired for various
luciferases. The invention provides compounds represented by
general formula (1) below. ##STR00001##
Inventors: |
INOUYE; Satoshi;
(Yokohama-shi, JP) ; SATO; Junichi; (Yokohama-shi,
JP) ; MIURA; Yuiko; (Yokohama-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JNC CORPORATION |
Tokyo |
|
JP |
|
|
Assignee: |
JNC CORPORATION
|
Family ID: |
50776858 |
Appl. No.: |
15/393315 |
Filed: |
December 29, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14244953 |
Apr 4, 2014 |
9567623 |
|
|
15393315 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6897 20130101;
C07D 487/04 20130101; C12Y 113/12007 20130101; C12Q 1/66
20130101 |
International
Class: |
C12Q 1/66 20060101
C12Q001/66; C12Q 1/68 20060101 C12Q001/68; C07D 487/04 20060101
C07D487/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 8, 2013 |
JP |
2013-080734 |
Claims
1-19. (canceled)
20. A method for measuring luminescence, which comprises contacting
f-coelenterazine and a luciferase, wherein said luciferase is
selected from the group consisting of (a), (b), and (c) below: (a)
a protein comprising the amino acid sequence of SEQ ID NO: 1; (b) a
protein comprising an amino acid sequence wherein 1 to 8 amino
acids are deleted, substituted, inserted and/or added in the amino
acid sequence of SEQ ID NO: 1 and having luminescence catalytic
activity with luciferin as a substrate; and (c) a protein
comprising an amino acid sequence having at least 95% identity to
the amino acid sequence of SEQ ID NO: 1 and having luminescence
catalytic activity with luciferin as a substrate,
21. The Method of claim 20, wherein the luciferase comprises the
amino acid sequence of SEQ ID NO: 1.
22. A method for assaying the activity of a target promoter
comprising: providing a polynucleotide encoding a luciferase as a
reporter gene and fusing said polynucleotide to said target
promoter to construct a vector; transforming said vector into a
host cell; and contacting said host cell with f-coelenterazine as a
luminescence substrate, wherein said luciferase encoded by said
polynucleotide is selected from the group consisting of (a), (b),
and (c) below: (a) a protein comprising the amino acid sequence of
SEQ ID NO: 1; (b) a protein comprising an amino acid sequence
wherein 1 to 8 amino acids are deleted, substituted, inserted
and/or added in the amino acid sequence of SEQ ID NO: 1 and having
luminescence catalytic activity with luciferin as a substrate; and
(c) a protein comprising an amino acid sequence having at least 95%
identity to the amino acid sequence of SEQ NO: I and having
luminescence catalytic activity with luciferin as a substrate.
23. The method of claim 22, wherein said luciferase comprises the
amino acid sequence of SEQ ID NO: 1,
24. The method of claim 22, wherein said polynucleotide encoding
said luciferase is a polynucleotide selected from the group
consisting of (a), (b), (c), and (d) below: (a) a polynucleotide
comprising the nucleotide sequence of SEQ ID NO: 2; (b) a
polynucleotide comprising a nucleotide sequence in which at least
one nucleotide is deleted, substituted, inserted and/or added in
the nucleotide sequence of SEQ ID NO: 2 and wherein said
polynucleotide encodes a protein having luminescence catalytic
activity with luciferin as a substrate; (c) a polynucleotide
comprising a nucleotide sequence having at least 90% identity to
the nucleotide sequence of SEQ ID NO: 2 and wherein said
polynucleotide encodes a protein having luminescence catalytic
activity with luciferin as a substrate; and (d) a polynucleotide
comprising a polynucleotide that hybridizes under stringent
conditions to a polynucleotide consisting of a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 2 and
encodes a protein having luminescence catalytic activity with a
Iticiferin as a substrate.
25. The method of claim 22, wherein said polynucleotide encoding
said luciferase comprises the nucleotide sequence of SEQ ID NO:
2.
26. A kit comprising: (i) f-coelenterazine; and (ii) a composition
selected from the group consisting of a luciferase, a
polynucleotide encoding said luciferase, a recombinant vector
comprising said polynucleotide encoding said luciferase, and a
transformant comprising said polynucleotide encoding said
luciferase, wherein said luciferase is selected from the group
consisting of (a), (b), and (c) below: (a) a protein comprising the
amino acid sequence of SEQ ID NO: 1; (b) a protein comprising an
amino acid sequence Wherein 1 to 8 amino acids are deleted,
substituted, inserted and/or added in the amino acid sequence of
SEQ ID NO: 1 and having luminescence catalytic activity with
luciferin as a substrate; and (c) a protein comprising an amino
acid sequence having at least 95% identity to the amino acid
sequence of SEQ ID NO: 1 and having luminescence catalytic activity
with luciferin as a substrate.
27. The kit of claim 26, wherein said luciferase comprises the
amino acid sequence of SEQ ID NO: 1.
28. The kit of claim 26, wherein said polynucleotide encoding said
luciferase is a polynucleotide selected from the group consisting
of (a), (b), (c) and (d) below: (a) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO: 2; (b) a polynucleotide
comprising a nucleotide sequence in which at least one nucleotide
is deleted, substituted, inserted and/or added in the nucleotide
sequence of SEQ ID NO: 2 and wherein said polynucleotide encodes a
protein having luminescence catalytic activity with luciferin as a
substrate; (c) a polynucleotide comprising a nucleotide sequence
having at least 90% identity to the nucleotide sequence of SEQ ID
NO: 2 and wherein said polynucleotide encodes a protein having
luminescence catalytic activity with luciferin as a substrate; and
(d) a polynucleotide comprising a polynucleotide that hybridizes
under stringent conditions to a polynucleotide consisting of a
nucleotide sequence complementary to the nucleotide sequence of SEQ
II) NO: 2 and encodes a protein having luminescence catalytic
activity with a luciferin as a substrate.
29. The kit of claim 26, wherein said polynucleotide encoding said
luciferase comprises the nucleotide sequence of SEQ II) NO: 2.
Description
TECHNICAL FIELD
[0001] The present invention relates to coelenterazine analogs, a
method for use thereof, and so on.
BACKGROUND ART
[0002] Bioluminescence is a phenomenon based on a chemical reaction
in vivo, which is called a luciferin (luminescence
substrate)-luciferase (enzyme that catalyzes the luminescence
reaction) reaction. Numerous studies including the identification
of luciferins or luciferases and the elucidation of the
luminescence mechanism in a molecular level have been performed for
a long time in the country and overseas.
[0003] It is known that many marine organisms utilize the compounds
having an imidazopyrazinone structure as a luminescence substrate
in the bioluminescence reaction.
[0004] Among the compounds, coelenterazine (CTZ) is known as the
luminescence substrate for Renilla luciferase, Oplaphorus
luciferase, Gaussia luciferase, etc., or also as the light-emitting
substrate of aequorin which is a photoprotein obtained from the
jellyfish. Coelenterazine is a compound commonly used as the
luminescence substrate for various luciferases and photoproteins,
and many findings of CTZ have been accumulated so far (Non-Patent
Documents 1 to 9).
##STR00002##
[0005] As the luminescence reaction system using coelenterazine as
a luminescence substrate proceeds only by the luminescence
substrate and molecular oxygen, the reporter assays using a
luciferase gene have been performed widely.
[0006] To date, approximately 50 coelenterazine analogs (CTZ
analogs) have been synthesized and the luminescence properties of
various luciferases have been studied using them as substrates
(Non-Patent Documents 6 to 10).
[0007] It is also shown that coelenterazine readily undergoes
oxidative degradation in an aqueous solution (Non-Patent Document
9).
[0008] Oplophorus luciferase is composed of proteins with molecular
weights of 35 kDa and 19 kDa. The domain that catalyzes the
luminescence reaction is found in the 19 kDa protein. In recent
years, a luminescence catalytic domain of 19 kDa having the amino
acid mutations has been reported and showed a higher luminescence
activity than that of native 19 kDa protein (Non-Patent Document
10).
[0009] In particular, Oplophorus luciferase and the 19 kD protein
which catalyzes the luminescence reaction showed broad substrate
specificity than other coelenterazine-type luciferases, indicating
that it is difficult to find coelenterazine analogs having 5 times
or higher luminescence activity than that of coelenterazine
(Non-Patent Documents 6 to 10). Furthermore, when coelenterazine is
applied to use for bioluminescence imaging, etc., it has also been
desired that coelenterazines are less susceptible to oxidative
degradation in an aqueous solution, i.e., a solution for the
luminescence reaction.
PRIOR ART REFERENCES
[0010] [Non-Patent Document 1] Teranishi K. (2007) Bioorg. Chem.
35, 82-111. [0011] [Non-Patent Document 2] Shimomura O. et al.
(1990) Biochem. J 270, 309-312. [0012] [Non-Patent Document 3]
Shimomura O. et al. (1988) Biochem. J. 251, 405-410. [0013]
[Non-Patent Document 4] Shimomura O. et al. (1989) Biochem. J. 261,
913-920. [0014] [Non-Patent Document 5] Inouye S. & Shimomura
O. (1997) Biochem. Biophys. Res, Commun. 233, 349-353 [0015]
[Non-Patent Document 6] Nakamura H. et al. (1997) Tetrahedron Lett.
38, 6405-6406. [0016] [Non-Patent Document 7] Wu C. et al. (2001)
Tetrahedron Lett. 42, 2997-3000. [0017] [Non-Patent Document 8]
Inouye S. & Sasaki S. (2007) Protein Express. Purif 56,
261-268. [0018] [Non-Patent Document 9] Inouye S. et al. (2013)
Protein Express. Purif. 88, 150-156. [0019] [Non-Patent Document
10] Hall M. P. et al. ACS Chem. Biol. 2012; 7: 1848-1857.
DISCLOSURE OF THE INVENTION
Problems to be Solved by the Invention
[0020] Coelenterazine acts as the luminescence substrate, which is
used in numerous luciferases from bioluminescent marine organisms
in common. No significant homology is detected in the primary
structures among these luciferases and their catalytic sites are
not clarified either. It is therefore impossible to precisely
design molecules of the luminescence substrate and the luciferase
protein and to predict a most suitable luciferin for the luciferase
protein. In order to find suitable substrates for individual
luciferases, it is necessary to screen them from coelenterazine
analogs.
[0021] Under the foregoing circumstances, there have been desired
coelenterazine analogs showing luminescence properties which are
different from those of known coelenterazine analogs.
Means for Solving the Problems
[0022] The present inventors have made extensive studies to solve
the problems described above and as a result, have found that novel
coelenterazine analogs having fluorine instead of hydroxy group on
the benzene ring at the position 2 and containing no hydroxy group
on the benzene ring at the position 6, of the coelenterazine
exhibit different luminescence properties for various luciferases
from those of known coelenterazine analogs. The present invention
has thus been accomplished.
[0023] More specifically, the present invention provides
coelenterazine analogs, a method for producing coelenterazine
analogs, kits, a luminescence method, etc.
[0024] [1] A compound represented by general formula (1) below:
##STR00003##
wherein:
[0025] R.sup.1 is hydrogen; a substituted or unsubstituted aryl; a
substituted or unsubstituted arylalkyl; a substituted or
unsubstituted aryl alkenyl; an alkyl which may optionally be
substituted with an alicyclic group; an alkenyl which may
optionally he substituted with an alicyclic group; an alicyclic
group; or a heterocyclic group.
[0026] [2] The compound according to [1] above, wherein, in general
formula (1), R.sup.1 is phenyl, p-hydroxyphenyl, benzyl,
.alpha.-hydroxybenzyl, p-hydroxybenzyl, phenylethyl, phenylvinyl,
cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl,
cyclohexylethyl, methyl, ethyl, propyl, 2-methylpropyl,
2-methylpropenyl, adamantylmethyl or thiophen-2-yl.
[0027] [3] The compound according to [2] above, wherein R.sup.1 is
benzyl, .alpha.-hydroxybenzyl or p-hydroxybenzyl in general formula
(1).
[0028] [4] The compound according to [3] above, wherein R.sup.1 is
benzyl in general formula (1).
[0029] [5] A method for producing a compound represented by general
formula (1) below:
##STR00004##
wherein, R.sup.1 is hydrogen; a substituted or unsubstituted aryl;
a substituted or unsubstituted arylalkyl; a substituted or
unsubstituted arylalkenyl; an alkyl which may optionally be
substituted with an alicyclic group; an alkenyl which may
optionally be substituted with an alicyclic group; an alicyclic
group; or a heterocyclic group, which comprises reacting a compound
represented by general formula (2) below:
##STR00005##
wherein, R.sup.1 is hydrogen; a substituted or unsubstituted aryl;
a substituted or unsubstituted arylalkyl; a substituted or
unsubstituted arylalkenyl; an alkyl which may optionally be
substituted with an alicyclic group; an alkenyl which may
optionally be substituted with an alicyclic group; an alicyclic
group; or a heterocyclic group, with a compound represented by
general formula (3) below:
##STR00006##
to give the compound represented by general formula (1),
[0030] [6] The method according to [5] above, wherein, in general
formula (1), R.sup.1 is phenyl, p-hydroxyphenyl, benzyl,
.alpha.-hydroxybenzyl, p-hydroxybenzyl, phenylethyl, phenylvinyl,
cyclopentyl, cyclopentylmethyl, cyclohexyl, cyclohexylmethyl,
cyclohexylethyl, methyl, ethyl, propyl, 2-methylpropyl,
2-methylpropenyi, adamantylmethyl or thiophen-2-yl.
[0031] [7] The method according to [6] above, wherein R.sup.1 is
benzyl, .alpha.-hydroxybenzyl or p-hydroxybenzyl in general formula
(1).
[0032] [8] The method according to [7] above, wherein R.sup.1 is
benzyl in general formula (1).
[0033] [9] A kit comprising:
[0034] (i) the compound according to any one of [1] to [4] above;
and,
[0035] (ii) at least one selected from a luciferase, a
polynucleotide encoding the luciferase, a recombinant vector
comprising the polynucleotide, and a transformant comprising the
polynucleotide.
[0036] [10] The kit according to [9] above, wherein said luciferase
is a 19 kDa protein of Oplophorus (Oplophorus sp.) luciferase.
[0037] [11] The kit according to [9] above, wherein said luciferase
is selected from the group consisting of (a) to (c) below:
[0038] (a) a protein comprising the amino acid sequence of SEQ ID
NO: 1,
[0039] (b) a protein comprising an amino acid sequence wherein 1 to
8 amino acids are deleted, substituted, inserted and/or added in
the amino acid sequence of SEQ ID NO: 1 and having a luminescence
catalytic activity with a luciferin as a substrate, and,
[0040] (c) a protein comprising an amino acid sequence having at
least 95% identity to the amino acid sequence of SEQ ID NO: 1 and
having a luminescence catalytic activity with a luciferin as a
substrate.
[0041] [12] The kit according to [9] above, wherein the
polynucleotide encoding said luciferase is a polynucleotide
selected from the group consisting of (a) to (d) below:
[0042] (a) a polynucleotide comprising a polynucleotide consisting
of the nucleotide sequence of SEQ ID NO: 2,
[0043] (b) a polynucleotide comprising a polynucleotide consisting
of a nucleotide sequence in which at least one nucleotide is
deleted, substituted, inserted and/or added in the nucleotide
sequence of SEQ ID NO: 2 and encoding a protein having a
luminescence catalytic activity with a luciferin as a
substrate,
[0044] (c) a polynucleotide comprising a polynucleotide consisting
of a nucleotide sequence having at least 90% identity to the
nucleotide sequence of SEQ ID NO: 2 and encoding a protein having a
luminescence catalytic activity with a luciferin as a substrate,
and,
[0045] (d) a polynucleotide comprising a polynucleotide that
hybridizes under stringent conditions to a polynucleotide
consisting of a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 2 and encodes a protein having a
luminescence catalytic activity with a luciferin as a
substrate.
[0046] [13] A method for luminescence, which comprises contacting
the compound according to any one of [1] to [4] above with a
luciferase.
[0047] [14] The method according to [13] above, wherein said
luciferase is a 19 kDa protein of Oplophorus (Oplophorus sp.)
luciferase.
[0048] [15] The method according to [13] above, wherein said
luciferase is a protein selected from the group consisting of (a)
to (c) below:
[0049] (a) a protein comprising the amino acid sequence of SEQ ID
NO: 1,
[0050] (b) a protein comprising an amino acid sequence wherein 1 to
8 amino acids are deleted, substituted, inserted and/or added in
the amino acid sequence of SEQ ID NO: 1 and having a luminescence
catalytic activity with a luciferin as a substrate, and,
[0051] (c) a protein comprising an amino acid sequence having at
least 95% identity to the amino acid sequence of SEQ ID NO: 1 and
having a luminescence catalytic activity with a luciferin as a
substrate.
[0052] [16] A method for assaying the activity of a sequence
associated with promoter regulation, which comprises using a
polynucleotide encoding a luciferase as a reporter gene and, as a
luminescence substrate, the compound according to any one of [1] to
[4] above.
[0053] [17] The method according to [16] above, wherein said
luciferase is a 19 kDa protein of Oplophorus (Oplophorus sp)
luciferase,
[0054] [18] The method according to [16] above, wherein said
luciferase is a protein selected from the group consisting of (a)
to (c) below:
[0055] (a) a protein comprising the amino acid sequence of SEQ ID
NO: 1,
[0056] (b) a protein comprising an amino acid sequence wherein 1 to
8 amino acids are deleted, substituted, inserted and/or added in
the amino acid sequence of SEQ ID NO: 1 and having a luminescence
catalytic activity with a luciferin as a substrate, and,
[0057] (c) a protein comprising an amino acid sequence having at
least 95% identity to the amino acid sequence of SEQ ID NO: 1 and
having a luminescence catalytic activity with a luciferin as a
substrate.
[0058] [19] The method according to [16] above, wherein the
polynucleotide encoding said luciferase is a polynucleotide
selected from the group consisting of (a) to (d):
[0059] (a) a polynucleotide comprising a. polynucleotid.e
consisting of the nucleotide sequence of SEQ ID NO: 2,
[0060] (b) a polynucleotide comprising a polynucleotide consisting
of a nucleotide sequence in which at least one nucleotide is
deleted, substituted, inserted and/or added in the nucleotide
sequence of SEQ ID NO: 2 and encoding a protein having a
luminescence catalytic activity with a luciferin as a
substrate,
[0061] (c) a polynucleotide comprising a polynucleotide consisting
of a nucleotide sequence having at least 90% identity to the
nucleotide sequence of SEQ ID NO: 2 and encoding a protein having a
luminescence catalytic activity with a luciferin as a substrate,
and,
[0062] (d) a polynucleotide comprising a polynucleotide that
hybridizes under stringent conditions to a polynucleotide
consisting of a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 2 and encodes a protein having a
luminescence catalytic activity with a luciferin as a
substrate.
Effects of the Invention
[0063] The present invention provides a coelenterazine analog
having luminescence properties to various luciferases, which are
different from those of known coelenterazine analogs. The
coelenterazine analog in a preferred embodiment of the invention
serves as a substrate suitable for the mutated 19 kDa protein
showing a higher activity than the 19 kDa protein of native
Oplophorus luciferase which shows broad substrate specificity. The
coelenterazine analog in a preferred embodiment of the invention is
highly stable in its aqueous solution, compared to
coelenterazine.
BRIEF DESCRIPTION OF DRAWINGS
[0064] FIG. 1 shows a plasmid map of the expression vector
pcDNA3-GLsp having a secretory signal sequence of Gaussia
luciferase.
[0065] FIG. 2 shows a plasmid map of pcDNA3-GLsp-nanolCaz.
[0066] FIG. 3 shows the luminescence patterns of recombinant
nanoKAZ with coelenterazine analogs by comparison to
coelenterazines.
BEST MODE FOR CARRYING OUT THE INVENTION
[0067] Hereinafter the present invention will be described in
detail.
1. Coelenterazine Analogs of the Invention
[0068] The present invention provides the compounds represented by
general formula (1) below (which are sometimes referred to as "the
coelenterazine analogs of the present invention" herein).
##STR00007##
In formula (1):
[0069] R.sup.1 is hydrogen; a substituted or unsubstituted aryl; a
substituted or unsubstituted arylalkyl; a substituted or
unsubstituted arylalkenyl; an alkyl which may optionally be
substituted with an alicyclic group; an alkenyl which may
optionally be substituted with an alicyclic group; an alicyclic
group; or a heterocyclic group.
[0070] The "substituted or unsubstituted aryl" of R.sup.1 is, for
example, an aryl having 1 to 5 substituents, or an unsubstituted
aryl. The substituent includes, for example, at least one selected
from the group consisting of a halogen (e.g. fluorine, chlorine,
bromine or iodine), hydroxy, an alkyl having 1-6 carbon atoms, an
alkoxyl having 1-6 carbon atoms, amino and a dialkylamino having
1-6 carbon atoms. In some embodiments of the present invention, the
substituent is hydroxy. Specific examples of the "substituted or
unsubstituted aryl" include phenyl, p-hydroxyphenyl, p-aminophenyl,
p-dimethylaminophenyl, etc., preferably, phenyl or p-hydroxyphenyl.
In some embodiments of the present invention, the "substituted or
unsubstituted aryl" is an unsubstituted aryl, e.g., phenyl.
[0071] The "substituted or unsubstituted arylalkyl" of R.sup.1 is,
for example, an arylalkyl having 7-10 carbon atoms of 1 to 5
substituents, or an arylalkyll having 7-10 carbon atoms. The
substituent includes, for example, a halogen (e.g. fluorine,
chlorine, bromine or iodine), hydroxy, an alkyl having 1-6 carbon
atoms, an alkoxyl having 1-6 carbon atoms, amino and a dialkylamino
having 1-6 carbon atoms. The "substituted or unsubstituted
arylalkyl" includes, for example, benzyl, a-hydroxybenzyl,
p-hydroxybenzyl, p-dimethylaminobenzyl or phenylethyl, and
preferably, benzyl, .alpha.-hydroxybenzyl, p-hydroxybenzyl or
phenylethyl. In some embodiments of the present invention, the
"substituted or unsubstituted arylalkyl" is benzyl.
[0072] The "substituted or unsubstituted arylalkenyl" of R.sup.1
is, for example, an arylalkenyl having 8-10 carbon atoms of 1 to 5
substituents, or an arylalkenyl having 8-10 carbon atoms. The
substituent includes, for example, a halogen (e.g. fluorine,
chlorine, bromine or iodine), hydroxy, an alkyl having 1-6 carbon
atoms, an alkoxyl having 1-6 carbon atoms, amino and a dialkylamino
having 1-6 carbon atoms. The "substituted or unsubstituted
arylalkenyl" includes, for example, phenylvinyl,
p-hydroxyphenylvinyl or p-dimethylaminophenylvinyl. In some
embodiments of the present invention, the "substituted or
unsubstituted arylalkenyl" is an unsubstituted arylalkenyl, e.g.,
phenylvinyl.
[0073] The "alkyl which may optionally be substituted with an
alicyclic group" of R.sup.1 includes, for example, an unsubstituted
straight or branched alkyl having 1-4 carbon atoms and a straight
or branched alkyl having 1-4 carbon atoms, which is substituted
with, e.g., 1-10 alicyclic groups. The alicyclic group includes,
for example, cyclohexyl, cyclopentyl, adamantyl, cyclobutyl and
cyclopropyl. Preferred examples of the alicyclic group are
cyclohexyl, cyclopentyl and adamantyl. The "alkyl which may
optionally be substituted with an alicyclic group" includes, for
example, methyl, ethyl, propyl, 2-methylpropyl, adamantylmethyl,
cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl,
cyclobutylmethyl and cyclopropylmethyl, and preferably, methyl,
ethyl, propyl, 2-methylpropyl, adamantylmethyl, cyclopentylmethyl,
cyclohexylmethyl and cyclohexylethyl. In some embodiments of the
present invention, the "alkyl which may optionally be substituted
with an alicyclic group" is a straight alkyl which may optionally
be substituted with an alicyclic group, e.g., methyl, ethyl,
propyl, adamantylmethyl, cyclopentylmethyl, cyclohexylmethyl and
cyclohexylethyl.
[0074] The "alkenyl which may optionally be substituted with an
alicyclic group" includes an unsubstituted straight or branched
alkenyl having 2-6 carbon atoms and a straight or branched alkenyl
having 2-6 carbon atoms, which is substituted with, e.g., 1-10
alicyclic groups. The alicyclic group includes, for example,
cyclohexyl, cyclopentyl, adamantyl, cyclobutyl and cyclopropyl.
Preferred examples of the alicyclic group are cyclohexyl,
cyclopentyl and adamantyl. The "alkenyl which may optionally be
substituted with an alicyclic group" includes, for example, vinyl,
1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl and
2-methylpropenyl, and preferably, 2-inethylpropenyl.
[0075] The "alicyclic group" of R.sup.1 includes, for example,
cyclohexyl, cyclopentyl, adamantyl, cyclobutyl and cyclopropyl.
Preferred examples of the alicyclic group include cyclopentyl and
cyclohexyl.
[0076] The "heterocyclic group" in R.sup.1 includes, for example, a
group derived from a 5- to 7-membered ring containing, in addition
to carbon atoms, 1 to 3 atoms selected from the group consisting of
N, O and S as the atoms constituting the ring and bonded via carbon
atoms, a group formed by fusing two or more of such rings and
bonded via carbon, and a group formed by fusing such a ring to a
benzene ring and bonding via carbon atoms. Examples of the
"heterocycli.c group" include thiophen-2-yl, 2-furanyl and
4-pyridyl. In some embodiments of the present invention, the
"heterocyclic group" is a heterocyclic group containing sulfur,
e.g., thiophen-2-yl.
[0077] In a preferred embodiment of the present invention, R.sup.1
is phenyl, p-hydroxyphenyl, benzyl, a-hydroxybenzyl,
p-hydroxybenzyl, phenylethyl, phenylvinyl, cyclopentyl,
cyclopentylmethyl, cyclohexyl, cyclohexy.lmethyl, cyclohexylethyl,
methyl, ethyl, propyl, 2-methylpropyl, 2-methylpropenyi,
adamantylmethyl or thiophen-2-yl.
[0078] In a more preferred embodiment of the present invention,
R.sup.1 is benzyl, The compound represented by general formula (1)
in which R' is benzyl is the compound shown by formula below
(sometimes referred to as "C6d-fcoelenterazine" or "C6df-CTZ"
herein).
##STR00008##
[0079] In an embodiment of the present invention, the
coelenterazine analogs exhibit luminescence properties different
from those of known coelenterazine analogs.
2. Method for Producing Coelenterazine Analog of the Invention
[0080] The compounds represented by general formula (1)
("coelenterazine analogs of the present invention") can be produced
as follows.
[0081] Specifically, the compounds represented by general formula
(2) below:
##STR00009##
wherein R' is defined as above, is reacted with the compound
represented by general formula (3) below:
##STR00010##
whereby the compounds represented by general formula (1) can be
produced.
[0082] The compounds represented by general formula (2) can be
produced by known methods. For instance, the compounds represented
by general formula (2) can be produced by the method described in
Kishi, Y. et al., Tetrahedron Lett,, 13, 2747-2748 (1972), or
Adamczyk, M. et al., Org. Prep. Proeed Int, 33, 477-485 (2001), or
a modification thereof More specifically, the compounds represented
by general formula (2) can be produced as follows. That is, first,
cyclization of substituted phenylglyoxal aldoximes and
glycinonitrile derivatives is carried out using a Lewis acid
catalyst to form the pyrazine oxides. Subsequently, the pyrazine
oxides are subjected to catalytic hydrogenation using a Raney Ni or
the like as a catalyst to produce the compounds. Alternatively, the
compounds represented by general formula (2) can be produced by
conducting the Suzuki-Miyaura coupling reaction between
2-amino-5-bromopyrazine derivatives and substituted phenylboronic
acids or substituted phenylboronic acid pinacol esters.
[0083] The compound represented by general formula (3) can be
produced by known methods. For example, the compound represented by
general formula (3) can be prepared, e.g., by the methods described
in Adamczyk, M. et al., Synth. Commun., 32, 3199-3205 (2002), or
Baganz, H. & May, H.-J. Chem. Ber., 99, 3766-3770 (1966) and
Baganz, H. & May, H.-J. Angew. Chem, hit. Ed. Eng., 5, 420
(1966), or their modifications. More specifically, the compound
represented by general formula (3) can be produced as follows. That
is, the compound represented by general formula (3) can be produced
either by reacting a substituted benzyl Grignard reagent with ethyl
diethoxyacetate at a low temperature (-78.degree. C)., or by
reacting an .alpha.-diazo-.alpha.'-substituted phenyl ketone with
tert-butyl hypochlorite in ethanol.
[0084] Herein, solvents used for the method for producing the
compounds of the present invention represented by general formula
(1) are not particularly limited and various solvents can be used.
Examples of such solvents include dioxane, tetrahydrofuran, ether,
methanol, ethanol, water, etc. These solvents can be used alone or
as an admixture thereof.
[0085] In the method for producing the compounds of the present
invention represented by general formula (1), the reaction
temperature and reaction time are not particularly limited and are,
for example, 0.degree. C. to 200.degree. C. for 1 to 96 hours, room
temperature to 150.degree. C. for 3 to 72 hours, or 60.degree. C.
to 120.degree. C. for 6 to 24 hours.
3. Protein of the Invention
[0086] The proteins of the present invention are mutated proteins
of the 19 kDa protein of native Opiophorus luciferase, which
comprise an amino acid sequence of a polypeptide having an activity
substantially equivalent to that of the protein comprising the
amino acid sequence of SEQ ID NO: 1.
[0087] The "activity substantially equivalent to that of the
protein comprising the amino acid sequence of SEQ ID NO: 1" is
intended to mean, for example, a luminescence catalytic activity
with a luciferin as a substrate (herein sometimes referred to as
"luminescence activity"), that is, the activity of catalyzing the
reaction that the luciferin (e.g., coelenterazines) is oxidized
with oxygen molecules to form oxyluciferin in its excited state.
The oxyluciferin formed in the excited state emits visible light to
decay to the ground state.
[0088] The activity or function can be determined, e.g., by the
method described in Inouye, S. & Shimomura, O. (1977) Biochem.
Biophys. Res. Commun. 233, 349-353. Specifically, the luminescence
catalytic activity can be determined by mixing the protein of the
present invention with luciferin to initiate the luminescence
reaction and measuring the activity using a luminometer. Devices
commercially available, e.g., Luminescencer-PSN AB2200
(manufactured by Atto Co., Ltd.) or Centro 960 luminometer
(manufactured by Berthold Inc.) may be used as the luminometer.
[0089] The luciferin used in the present invention may be any
luciferin, so long as it is a luciferin which serves as a substrate
for the protein of the present invention. Specifically, the
luciferin used in the present invention includes coelenterazines
having an imidazopyrazinone ring as the basic skeleton.
[0090] The coelenterazines are intended to mean coelenterazine or
analogs thereof (e.g., the coelenterazine analogs of the present
invention). The coelenterazines can be synthesized by known methods
or are commercially available. The method for producing the
coelenterazine analogs of the present invention is as described
hereinabove.
[0091] The "luminescence catalytic activity with a luciferin as a
substrate" is preferably the luminescence catalytic activity with a
coelenterazine as a substrate. The "luminescence catalytic activity
with a coelenterazine as a substrate" preferably refers to the
luminescence catalytic activity with the coelenterazine analog of
the present invention as a substrate. More preferably, the
"luminescence catalytic activity with the coelenterazine analog of
the present invention as a substrate" refers to the luminescence
catalytic activity showing a relative maximum luminescence
intensity 5 times or higher as compared to coelenterazine, when the
coelenterazine analog of the present invention is used as a
substrate. Most preferably, when the coelenterazine analog of the
present invention is used as a substrate, it is the luminescence
catalytic activity Showing a relative maximum luminescence
intensity 5 times or higher as compared to coelenterazine that
provides a continuous luminescence. In terms of the relative
maximum luminescence intensity, "5 times or higher" is, for
example, 5 to 20 times, 5 to 15 times, 5 to 14 times, 5 to 13
times, 5 to 12 times or 5 to 11 times. The time period for
continuous luminescence in the "continuous luminescence" is, for
example, 1 minute to 120 minutes, 1 minute to 60 minutes, 1 minute
to 30 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, 1
minute to 5 minutes or 1 minute to 3 minutes.
[0092] The "protein which comprise an amino acid sequence of a
polypeptide having an activity substantially equivalent to that of
the protein comprising the amino acid sequence of SEQ ID NO: 1" is
a protein selected from the group consisting of (a) to (c)
below.
[0093] (a) a protein comprising the amino acid sequence of SEQ ID
NO: 1,
[0094] (b) a protein comprising an amino acid sequence wherein 1 to
8 amino acids are deleted, substituted, inserted and/or added in
the amino acid sequence of SEQ ID NO: 1 and having the luminescence
catalytic activity with a luciferin as a substrate, and,
[0095] (c) a protein comprising an amino acid sequence having at
least 95% identity to the amino acid sequence of SEQ ID NO: 1 and
having the luminescence catalytic activity with a luciferin as a
substrate.
[0096] As used herein, the "1 to 8 amino acids are deleted,
substituted, inserted and/or added" is intended to mean that
deletion, substitution, insertion and/or addition of 1 to 8 amino
acid residues may occur at any optional and 1 to 8 positions of in
the same amino acid sequence.
[0097] The range of "1 to 8" in the "amino acid sequence wherein 1
to 8 amino acids are deleted, substituted, inserted and/or added"
is, for example, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1
to 4, 1 to 3, 1 to 2, or 1. In general, the less the number of
amino acids deleted, substituted, inserted and/or added, the more
preferable. in the deletion, substitution, insertion and addition
of the amino acid residues described above, two or more may occur
concurrently. Such domains can be acquired using site-directed
mutagenesis described in "Sambrook J. et al., Molecular Cloning: A
Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory
Press (2001)," "Ausbel F. M. et al., Current Protocols in Molecular
Biology, Supplement 1-38, John Wiley and Sons (1987-1997) ," "Nuc.
Acids. Res., 10, 6487 (1982)," "Proc. Natl. Acad. Sci. USA, 79,
6409 (1982)," "Gene, 34, 315 (1985)," "Nuc. Acids. Res., 13, 4431
(1985)," "Proc. Natl. Acad. Sci. USA, 82, 488 (1985)," etc.
[0098] Examples of mutually substitutable amino acid residues are
given below. [0099] The amino acid residues in the same group can
be mutually substituted. [0100] Group A: leucine, isoleucine,
norleucine, valine, norvaline, alanine, 2-aminobutanoic acid,
methionine, o-methylserine, t-butylglycine, t-butylalanine and
cyclohexylalanine; [0101] Group B: aspartic acid, glutamic acid,
isoaspartic acid, isoglutamic acid, 2-aminoadipic acid and
2-aminosuberic acid; [0102] Group C: asparagine and glutamine;
[0103] Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic
acid and 2,3-diaminopropionic acid; [0104] Group E: proline,
3-hydroxyproline and 4-hydroxyproline; [0105] Group F: serine,
threonine and homoserine; and, [0106] Group G: phenylalanine and
tyrosine.
[0107] In the amino acid sequence wherein 1 to 8 amino acids are
deleted, substituted, inserted and/or added in the amino acid
sequence of SEQ ID NO: 1, it is preferred that none of the
following amino acids are deleted or substituted in the amino acid
sequence of SEQ ID NO: 1; Glu at position 6, Arg at position 13,
Leu at position 20, Leu at position 29, Asn at position 35, Arg at
position 45, Ile at position 46, Ile at position 56, Asp at
position 70, Gin at position 74, Lys at position 77, Val at
position 92, Glu at position 117, Lys at position 126, Ile at
position 140 and Arg at position 168.
[0108] As used herein, the range of "at least 95%" in the "amino
acid sequence having at least 95% identity" is, for example, 95% or
more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or
more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more,
99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more. In
general, the numerical value indicating the identity above is
preferably higher. The identity between amino acid sequences or
nucleotide sequences can be determined using a sequencing program
such as BLAST (see, e.g., Altzshul S. F. et al., J. Mol. Biol. 215,
403 (1990), etc.). When BLAST is used, the default parameters for
the respective programs are employed.
[0109] The protein of the present invention may be a protein
encoded by the polynucleotide of the invention later described.
[0110] Preferably, the protein of the present invention is a
protein selected from the group consisting of (a) to (c) below.
[0111] (a) a protein comprising the amino acid sequence of SEQ ID
NO: 1,
[0112] (b) a protein comprising an amino acid sequence wherein 1 to
4 amino acids are deleted, substituted, inserted and/or added in
the amino acid sequence of SEQ ID NO: 1 and having the luminescence
catalytic activity with a luciferin as a substrate, and,
[0113] (c) a protein comprising an amino acid sequence having at
least 98% identity to the amino acid sequence of SEQ ID NO: 1 and
having the luminescence catalytic activity with a :luciferin as a
substrate.
[0114] More preferably, the protein of the present invention is a
protein comprising a polypeptide consisting of the amino acid
sequence of SEQ ID NO: 1.
[0115] The protein of the invention may further contain an
additional peptide sequence at the N terminus and/or the C
terminus, preferably at the N terminus. The additional peptide
sequence includes, for example, at least one peptide sequence
selected from the group consisting of a peptide sequence for
promoting translation, a peptide sequence for purification, a
secretory signal peptide sequence, a peptide sequence for
expressing the protein of the invention as a soluble protein and an
epitope sequence capable of recognizing an antibody. The additional
peptide sequence is preferably a peptide sequence for purification
and/or a secretory signal peptide sequence. In another preferred
embodiment of the present invention, the additional peptide
sequence is at least one selected from the group consisting of a
peptide sequence for purification, a secretory signal peptide
sequence, and a sequence for expressing the protein of the present
invention as a soluble protein.
[0116] The protein of the present invention may further contain a
linker sequence for restriction enzyme sites.
[0117] Peptide sequences used in the art can be used as the peptide
sequence for promoting translation. The peptide sequence for
promoting translation includes, fbr example, a TEE sequence.
[0118] Peptide sequences used in the art can be used as the peptide
sequence for purification. The peptide sequence for purification
includes, fbr example, a histidine tag sequence having a
consecutive amino acid sequence of at least 4 histidine residues,
preferably at least 6 histidine residues, an amino acid sequence
with a binding domain of glutathione S-transferase into
glutathione, the amino acid sequence of Protein A and an avidin tag
sequence.
[0119] The secretory signal peptide is intended to mean a peptide
region which plays a role of transporting a protein bound to the
secretory signal peptide across a cell membrane. Amino acid
sequences of such secretory signal peptides and nucleic acid
sequences encoding the same are well known in the art and reported
(see, e.g., von Heijine G (1988) Biochim. Biohys. Acta 947:
307-333, von Heijine G (1990) J. Membr. Biol. 115: 195-201).
Specific examples of the secretory signal peptides include the
secretory signal peptide from outer membrane protein A of
Escherichia coli (OmpA) (Ghrayeb, J. et al. (1984) EMBO J.
3:2437-2442), the secretory signal peptide from Vibrio
cholerae-derived cholera toxin and the secretory signal peptide of
Gaussia luciferase used in EXAMPLES later described.
[0120] The peptide sequence for expressing the protein of the
present invention as a soluble protein includes, for example, a
polypeptide represented by formula (Z).sub.n (ZZ domain in
particular). Amino acid sequences of the polypeptide represented by
formula (Z).sub.n and nucleic acid sequences encoding the same
include those described in Japanese Patent Application KOKAI No.
2008-99669, and the like.
[0121] As the linker sequence for a restriction enzyme site, there
can be used peptide sequences used in the art.
[0122] In some embodiments of the present invention, the protein
includes a protein comprising a polypeptide consisting of the amino
acid sequence of SEQ II) NO: 6 or SEQ ID NO: 7.
[0123] The method for obtaining the protein of the invention is not
particularly limited. The protein of the invention may be a protein
synthesized by chemical synthesis, or a recombinant protein
produced by a genetic engineering technique. If the protein of the
invention is to be chemically synthesized, synthesis may be carried
out by, for example, the Fmoc (fluorenylmethyloxycarbonyl) method
or the tBoc (t-butyloxycarbonyl) method. Also, peptide synthesizers
available from, for example, Advanced ChemTech, PerkinElmer,
Pharmacia, Protein Technology Instrument, Synthecell-Vega,
PerSeptive, Shimadzu Corporation, etc. may be used for chemical
synthesis. Where the protein of the invention is to he produced by
a genetic engineering technique, the protein may be produced by a
conventional genetic recombination technique. More specifically,
the protein of the invention may be produced by inserting a
polynucleotide (e.g., a DNA) encoding the protein of the invention
into a suitable expression system.
4. Polynucleotide of the Invention
[0124] The present invention also provides a polynucleotide
encoding the protein of the invention described above. The
polynucleotide of the invention may be any polynucleotide as long
as it comprises a nucleotide sequence encoding the protein of the
invention, although a DNA is preferred. Examples of DNA include
genomic DNA, genomic DNA library, cellular or tissue cDNA, cellular
or tissue cDNA library, synthetic DNA, etc. Vectors used in the
libraries are not particularly limited and may be any of
bacteriophages, plasmids, cosmids, phagemids, etc. Also, these
vectors may be amplified directly by a reverse transcription
polymerase chain reaction (hereinafter abbreviated as RT-PCR) using
the total RNA or mRNA fraction prepared from the cell or tissue
described above.
[0125] Specifically, the polynucleotide of the invention is any one
of the polynucleotides selected from the group consisting of (a) to
(d) below.
[0126] (a) a polynucleotide comprising a polynucleotide consisting
of the nucleotide sequence of SEQ ID NO: 2,
[0127] (b) a polynucleotide comprising a polynucleotide encoding a
protein consisting of a nucleotide sequence in which at least one
nucleotide is deleted, substituted, inserted and/or added in the
nucleotide sequence of SEQ ID NO: 2 and having the luminescence
catalytic activity with a luciferin as a substrate,
[0128] (c) a polynucleotide comprising a polynucleotide encoding a
protein consisting of a nucleotide sequence having at least 90%
identity to the nucleotide sequence of SEQ ID NO: 2 and having the
luminescence catalytic activity with a. luciferin as a substrate,
and,
[0129] (d) a polynucleotide comprising a polynucleotide that
hybridizes under stringent conditions to a polynucleotide
consisting of a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 2 and encoding a protein having the
luminescence catalytic activity with a luciferin as a
substrate.
[0130] As used herein, the "luminescence catalytic activity with a
luciferin as a substrate" is defined as above.
[0131] The "nucleotide sequence in which at least one nucleotide is
deleted, substituted, inserted and/or added" is intended to mean
that deletion, substitution, insertion and/or addition of one or
more nucleotides may occur at any optional and one or more
positions in the same nucleotide sequence.
[0132] The range of "at least one" in the "nucleotide sequence in
which at least one nucleotide is deleted, substituted, inserted
and/or added" is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1
to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to
2, or 1. In general, the less the number of nucleotides deleted,
substituted, inserted and/or added, the more preferable. In the
deletion, substitution, insertion and addition of the nucleotides
described above, two or more may occur at the same time. Such
domains can be acquired using site-directed mutagenesis described
in "Sambrook J. et al., Molecular Cloning: A Laboratory Manual,
Third Edition, Cold Spring Harbor Laboratory Press (2001)," "Ausbel
F. M. et al., Current Protocols in Molecular Biology, Supplement
1-38, John Wiley and Sons (1987-1997)," "Nuc. Acids. Res., 10, 6487
(1982)," "Proc. Natl. Acad. Sci. USA, 79, 6409 (1982)," "Gene, 34,
315 (1985)," "Nuc. Acids. Res., 13, 4431 (1985)," "Proc. Natl.
Acad. Sci. USA, 82, 488 (1985)," etc.
[0133] A polynucleotide, in which one or more nucleotides are
deleted, substituted, inserted and/or added in a certain nucleotide
sequence, can he obtained by using a site-specific mutagenesis
technique (cf., e.g., Gotoh, T. et al., Gene 152, 271-275 (1995),
Zoller, M. J., and Smith, M., Methods Enzymoi. 100, 468-500 (1983),
Kramer, W. et al., Nucleic Acids Res. 12, 9441-9456 (1984), Kramer
W, and Fritz H. J., Methods. Enzymol. 154, 350-367 (1987), Kunkel,
T. A., Proc. Natl. Acad. Sci. USA. 82, 488-492 (1985) and Kunkel,
Methods Enzymol. 85, 2763-2766 (1988)), methods using amber
mutation (cf., e.g., the gapped duplex method, Nucleic Acids Res.
12, 9441-9456 (1984)), etc.
[0134] Alternatively, such mutation may be introduced into the
polynucleotide by PCR using a set of primers bearing on the
respective 5' ends a sequence in which the target mutation
(deletion, addition, substitution and/or insertion) has been
introduced (see, e.g., Ho S. N. etal., Gene 77, 51 (1989),
etc.).
[0135] Also, the polynucleotide encoding a partial protein
fragment, which is one type of deletion mutants, can be obtained
using as the primers an oligonucleotide having a sequence which
matches the nucleotide sequence at the 5' end of the region
encoding the partial fragment to be produced in the polynucleotide
encoding the protein and an oligonucleotide having a sequence
complementary to the nucleotide sequence at the 3' end thereof, and
performing PCR using a polynucleotide encoding the protein as a
template.
[0136] The range of "90% or more" in the "nucleotide sequence
having at least 90% identity" is, for example, 90% or more, 91% or
more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or
more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2%
or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or
more, 99.7% or more, 99.8% or more, or 99.9% or more. In general,
the greater the numerical value of the identity above, the more
preferable. The identity between amino acid sequences or nucleotide
sequences can be determined using a sequencing program such as
BLAST (see, e.g., Altzshul, S. F. et aid. Mol. Biol., 215, 403
(1990), etc.) or the like, When BLAST is used, the default
parameters for the respective programs are employed.
[0137] That "polynucleotide that hybridizes under stringent
conditions" is intended to mean a polynucleotide (e.g., DNA) which
is obtained by, for example, colony hybridization, plaque
hybridization or Southern hybridization using as a probe all or
part of the polynucleotide consisting of a nucleotide sequence
complementary to the nucleotide sequence of SEQ ID NO: 2.
Specifically, the polynucleotide is a polynucleotide which can be
identified by performing hybridization at 65.degree. C. in the
presence of 0.7 to 1.0 mol/L NaCl using a filter on which the
polynucleotide from a colony or plaque is immobilized, then washing
the filter at 65.degree. C. with an SSC (saline-sodium citrate)
solution having a concentration of with 0.1- to 2 times (1.times.
SSC solution is composed of 150 mmol/L sodium chloride and 15
mmol/L sodium citrate).
[0138] Hybridization may be performed in accordance with
modifications of the methods described in experimental manuals,
e.g., Sambrook J. et al., Molecular Cloning: A Laboratory Manual,
Third Edition, Cold Spring Harbor Laboratory Press (2001), Ausbel
F. M. et al., Current Protocols in Molecular Biology, Supplement
1-38, John Wiley and Sons (1987-1997), Glover D. M. and Hames B.
D., DNA Cloning 1: Core Techniques, A practical Approach, Second
Edition, Oxford University Press (1995), etc.
[0139] The "stringent conditions" may refer to any of less
stringent conditions, moderately stringent conditions and highly
stringent conditions. The "less stringent conditions" are, for
example, the conditions under 5.times.SSC, 5.times. Denhardt's
solution, 0.5% (w/v) SDS and 50% (v/v) formamide at 32.degree. C.
The "moderately stringent conditions" are, for example, the
conditions under 5.times.SSC, 5.times. Denhardt's solution, 0.5%
(w/v) SDS and 50% (v/v) forinamide at 42.degree. C. The "highly
stringent conditions" are, for example, the conditions under
5.times.SSC, 5.times. Denhardt's solution, 0.5% (w/v) SIDS and 50%
(v/v) formamide at 50.degree. C. The more stringent the conditions
are, the higher the complementarity required for double strand
formation. Specifically, for example, under these conditions, a
polynucleotide (e.g., a DNA) of higher homology is expected to be
obtained efficiently at higher temperatures, although multiple
factors are involved in hybridization stringency, including
temperature, probe concentration, probe length, ionic strength,
time and base concentration, etc.; those skilled in the art may
appropriately choose these factors to realize a similar
stringency.
[0140] Where a kit commercially available is used for the
hybridization, for example, Alkphos Direct Labeling Reagents
(manufactured by Amersham Pharmacia) may be used. In this case,
incubation with a labeled probe is performed overnight in
accordance with the protocol attached to the kit, the membrane is
then washed with a primary wash buffer containing 0.1% (w/v) SDS at
55.degree. C. Thus, the hybridized DNA can be detected.
[0141] Other polynucleotides that are capable of hybridization
include, as calculated by a sequencing program such as BLAST or the
like using the default parameters. DNAs having an identity of
approximately 60% or more, 65% or more, 70% or more, 75% or more,
80% or more, 85% or more, 88% or more, 90% or more, 92% or more,
95% or more, 97% or more, 98% or more, 99% or more, 99.3% or more,
99.5% or more, 99.7% or more, 99.8% or more, or 99.9% or more, to
the polynucleotide encoding the polynucleotide consisting of the
nucleotide sequence of SEQ ID NO: 2. The identity between
nucleotide sequences can be determined using the method described
above,
[0142] In a preferred embodiment of the invention, the
polynucleotide is a polynucleotide selected from the group
consisting of (a) to (d) below.
[0143] (a) a polynucleotide comprising a polynucleotide consisting
of the nucleotide sequence of SEQ ID NO: 2,
[0144] (b) a polynucleotide comprising a polynucleotide consisting
of a nucleotide sequence in which 1 to 20 nucleotides are deleted,
substituted, inserted and/or added in the nucleotide sequence of
SEQ ID NO: 2 and encoding a protein having the luminescence
catalytic activity with a luciferin as a substrate,
[0145] (c) a polynucleotide comprising a polynucleotide consisting
of a nucleotide sequence having at least 95% identity' to the
nucleotide sequence of SEQ ID NO: 2 and encoding a protein having
the luminescence catalytic activity with a luciferin as a
substrate, and,
[0146] (d) a polynucleotide comprising a polynucleotide that
hybridizes under stringent conditions to a polynucleotide
consisting of a nucleotide sequence complementary to the nucleotide
sequence of SEQ ID NO: 2 and encodes a protein having the
luminescence catalytic activity with a luciferin as a
substrate.
[0147] In a still preferred embodiment of the invention, the
polynucleotide is a polynucleotide selected from the group
consisting of (a) to (c) below.
[0148] (a) a polynucleotide comprising a polynucleotide consisting
of the nucleotide sequence of SEQ II) NO: 2,
[0149] (b) a polynucleotide comprising a polynucleotide consisting
of a nucleotide sequence in which at least one nucleotide is
deleted, substituted, inserted and/or added in the nucleotide
sequence of SEQ ft) NO: 2 and encoding a protein having the
luminescence catalytic activity with a luciferin as a
substrate,
[0150] (c) a polynucleotide comprising a polynucleotide consisting
of a nucleotide sequence having at least 98% identity to the
nucleotide sequence of SEQ ID NO: 2 and encoding a protein having
the luminescence catalytic activity with a luciferin as a
substrate.
[0151] In a particularly preferred embodiment of the invention, the
polynucleotide is a polynucleotide comprising a polynucleotide
consisting of the nucleotide sequence of SEQ ID NO: 2.
[0152] The polynucleotide of the present invention may further
contain a polynucleotide comprising a polynucleotide encoding an
additional peptide sequence. The additional peptide sequence
includes, for example, at least one peptide sequence selected from
the group consisting of a peptide sequence for promoting
translation, a peptide sequence for purification, a secretory
signal peptide sequence, a peptide sequence for expressing the
fusion protein of the invention as a soluble protein and an epitope
sequence capable of recognizing an antibody.
[0153] The polynucleotide of the present invention may further
contain a polynucleotide encoding the linker sequence for
restriction enzyme sites.
[0154] Polynucleotides comprising polynucleotides encoding the
peptide sequences for promoting translation which are used in the
art can be employed as the polynucleotide comprising a
polynucleotide encoding the peptide sequence for promoting
translation. Examples of the peptide sequence for promoting
translation include those described above.
[0155] Polynucleotides comprising nucleotide sequences encoding the
peptide sequences for purification which are used in the art can be
employed as the polynucleotide comprising a nucleotide sequence
encoding the peptide sequence for purification. Examples of the
peptide sequence for purification include those described
above.
[0156] Polynucleotides comprising nucleic acid sequences encoding
secretory signal peptides which are known in the art can be
employed as the polynucleotide encoding the secretory signal
peptide. Examples of the secretory signal peptide include those
described above.
[0157] Polynucleotides comprising nucleic acid sequences encoding
peptides for expressing as soluble proteins peptide sequences which
are known in the art can be employed as the polynucleotide encoding
the peptide sequence for expressing the protein of the invention as
a soluble protein. Examples of the peptide for expressing the
protein of the invention as a soluble protein include those
described above.
[0158] Polynucleotides comprising nucleotide sequences encoding
linker sequences which are used in the art can be employed as the
polynucleotide encoding the linker sequence for restriction enzyme
sites.
[0159] In some embodiments of the invention, the polynucleotide is
a polynucleotide comprising a polynucleotide consisting of the
nucleotide sequence of SEQ ID NO: 5.
5. Method for Producing Calcium-Binding Photoprotein
[0160] The calcium-binding photoprotein of the invention can be
produced or regenerated by contacting the coelenterazine analog of
the invention with the apoprotein of the calcium-binding
photoprotein thereby to obtain the calcium-binding
photoprotein.
[0161] As used herein, the term "contact" is intended to mean that
the coelenterazine analog of the invention and the apoprotein of
the calcium-binding photoprotein are allowed to exist in the same
reaction system, and includes, for example, the apoprotein of the
calcium-binding photoprotein being added to a container charged
with the coelenterazine analog of the invention, the coelenterazine
analog of the invention being added to a container charged with the
apoprotein of the calcium-binding photoprotein, and the
coelenterazine analog of the invention being mixed with the
apoprotein of the calcium-binding photoprotein. In one embodiment
of the present invention, the contact is carried out at a low
temperature in the presence of a reducing agent (e.g.,
mercaptoethanol or dithiothreitol) and oxygen. More specifically,
the photoprotein of the present invention can be produced or
regenerated. by the methods described in, e.g., Shimomura, O. et
al. Biochem. J. 251, 405-410 (1988) and Shimomura, O. et al.
Biochem. J. 261, 913-920 (1989). The calcium-binding photoprotein
of the present invention is present in such a state that a complex
is formed between the peroxide of the coelenterazine analog
generated from the coelenterazine analog of the invention and
molecular oxygen and the apoprotein. Calcium ions are bound to the
complex above to generate instantaneous luminescence and form the
coelenteramide analog, which is the oxide of the coelenterazine
analog, and carbon dioxide. The complex above is sometimes referred
to as "the calcium-binding photoprotein of the present
invention."
[0162] The apoprotein used to produce the calcium-binding
photoprotein of the present invention may be collected from natural
sources or produced by genetic engineering. In addition, the
apoprotein may be any mutant which amino acid sequence is mutated
from the natural sequence by gene recombination technology as long
as it is capable of producing the calcium-binding photoprotein.
Specific examples of the apoprotein include apoaequorin,
apoclytin-I, apoclytin-II, apobelin, apomitrocomin, apomineopsin,
apobervoin, and mutated proteins thereof In some embodiments of the
present invention, the apoprotein is apoaequorin, apobelin,
apoclytin-I, apoclytin-lI or mitrocomin, or mutated proteins, etc.,
preferably, apoaequorin or its mutated protein. These apoproteins
may be produced by known methods or a modification of these
methods. Alternatively, various products are commercially available
from NC Corporation and these commercial products may be used as
well.
6. Use of Coelenterazine Analogs of the Invention
Use of Detection Marker as Luminescence Substrate by
Luminescence
[0163] The coelenterazine analog of the present invention can be
used as a detection marker by luminescence. The coelenterazine
analog of the present invention can be used as a luminescence
substrate for the detection marker to detect a target analyte in
immunoassay, hybridization assay, etc.
[0164] The detection marker of the invention may also be used, for
example, by expressing a luciferase or the apoprotein of
calcium-binding photoprotein (hereinafter sometimes referred to as
"luciferase, etc.") as a fusion protein to the target protein,
inserting the fusion protein into a cell by means of
microinjection, etc. and contacting the cell with the
coelenterazine analog of the invention, thereby to determine
distribution of the target protein described above. As used herein,
the term "contact" is intended to mean that a cell and the
coelenterazine analog of the invention are allowed to be present in
the same culture system or reaction system, and includes, for
example, the coelenterazine analog of the invention being added to
a culture container charged with a cell, a cell being mixed with
the coelenterazine analog of the invention, and a cell being
cultured in the presence of the coelenterazine analog of the
invention. Such distribution of the target protein, etc. may also
be determined by utilizing a detection method such as luminescence
imaging, etc. In addition to the insertion into a cell by means of
microinjection, etc., the luciferase, etc. may also be expressed in
a cell and provided for use.
[0165] The luciferase may be collected from natural sources or
produced by genetic engineering. Furthermore, the luciferase may be
any mutant in which amino acid sequence is mutated from the natural
sequence by gene recombination technology. The luciferase may also
be a domain that catalyzes luminescence of the native luciferase or
its mutated protein. The luciferase is, for example, a luciferase
(Oplophorus luciferase) derived from Oplophorus sp. (e.g.,
Oplophorus gracilorostris), a domain (19 kDa protein) that
catalyzes luminescence of Oplophorus luciferase, a luciferase
(Renilla luciferase) derived from Renilla sp. (e.g., Renilla
renifonnis or Renilla muelleri) and their mutated proteins. These
luciferases and their mutated proteins (e.g., the protein of the
present invention) can be produced by the method described in,
e.g., Shimomura et al. (1988) Biochem. J. 251, 405-410, Shimomura
et al. (1989) Biochem. J. 261, 913-920, or Shimomura et al. (1990)
Biochem. J. 270, 309-312, or a modification thereof Alternatively,
as various products are commercially available from JNC
Corporation, Wako Pure Chemicals Industries, Promega Corporation,
etc., these products may he used as well. The method for producing
the protein of the present invention is as described
hereinabove.
[0166] The apoprotein is as described above.
[0167] The luciferase, etc. used is preferably the 19 kDa protein
of Oplophorus luciferase and its mutated proteins (e.g., the
protein of the present invention), and more preferably, the protein
of the present invention.
[0168] In Oplophorus luciferase, the nucleotide sequence and amino
acid sequence of the 19 kDa protein of luciferase from Oplophorus
gracilorostris are shown in SEQ ID NO: 8 and SEQ ID NO: 9,
respectively.
Use of Rrportr Protein as Luminescence Substrate
[0169] The coelenterazine analog of the present invention may also
be used as a luminescence substrate for reporter proteins to
determine the activity (transcription activity) of a sequence
engaged in promoter regulation. The polynucleotide encoding the
luciferase, etc. is fused to a target promoter or other expression
control sequence (e.g., an enhancer, etc.) to construct a vector.
The vector is transformed to a host cell. The coelenterazine analog
of the present invention is brought in contact with the host cell.
By detecting the luminescence from the luciferin, etc., the
activity of the target promoter or other expression control
sequence can be determined. As used herein, the term "contact" is
intended to mean that a host cell and the coelenterazine analog of
the invention are allowed to be present in the same culture system
or reaction system, and includes, for example, the coelenterazine
analog of the invention being added to a culture container charged
with a host cell, a host cell being mixed with the coelenterazine
analog of the invention, a host cell being cultured in the presence
of the coelenterazine analog of the invention, and the like.
[0170] The luciferin, etc. is as described in the section entitled
"Use of Detection Marker as Luminescence Substrate by
Luminescence."
[0171] The luciferin used is preferably the 19 kDa protein of
Qplophorus luciferase and its mutated proteins (e.g., the protein
of the present invention), and more preferably, the protein of the
present invention.
[0172] The polynucleotide of the present invention y preferably be
used as a reporter gene, as described above.
Material for Amusement Supplies
[0173] The coelenterazine analog of the present invention can be
advantageously used as a luminescence substrate when the
luciferase, etc. is used as a luminescence substrate in materials
for amusement supplies. Examples of the amusement supplies include
luminescent bubble soap, luminescent ice, luminescent candies,
luminescent paints, etc. The amusement supplies of the invention
can be prepared in a conventional manner.
[0174] The luciferase, etc. used is preferably the 19 kDa protein
of Oplophorus luciferase and its mutated proteins (e.g., the
protein of the present invention), and more preferably, the protein
of the present invention.
Bioluminescence Resonance Energy Transfer (BRET) Method
[0175] The coelenterazine analog of the present invention can be
used as a luminescence substrate for the luciferase, etc., in the
method of analyses such as an analysis of biological functions,
measurement of enzyme activities, etc., based on the principle of
intermolecular interactions by the bioluminescence resonance energy
transfer (BRET) method.
[0176] For example, using the luciferase, etc. and the
coelenterazine analog of the present invention as donors and a
fluorescent substance (e.g., an organic compound or a fluorescent
protein) as an acceptor, the interactions between the donors and
acceptor can be detected by causing bioluminescence resonance
energy transfer (BRET) between them.
[0177] In an embodiment of the present invention, the organic
compound used as an acceptor is Hoechst 3342, Indo-1, DAP1, etc. In
another embodiment of the present invention, the fluorescent
protein used as an acceptor is a green fluorescent protein (GFP), a
blue fluorescent protein (BFP), a mutant GFP fluorescent protein,
phycobilin, etc.
[0178] In a preferred embodiment of the present invention, the
physiological functions to be analyzed include an orphan receptor
(in particular, a G-protein conjugated receptor), apoptosis,
transcription regulation by gene expression, etc. In a preferred
embodiment of the present invention, the enzyme to be analyzed is
protease, esterase, kinase, or the like.
[0179] Analysis of the physiological functions by the BRET method
may be performed by publicly known methods, for example, by a
modification of the method described in Biochem. J. 2005, 385,
625-637 or Expert Opin. Ther Tarets, 2007 11: 541-556. Assay for
the enzyme activity may also be performed by publicly known
methods, for example, by a modification of the method described in
Nature Methods 2006, 3:165-174 or Biotechnol J. 2008,
3:311-324.
[0180] The luciferase, etc. used is preferably the 19 kDa protein
of Oplophorus luciferase and its mutated proteins (e.g., the
protein of the present invention), and more preferably, the protein
of the present invention.
7. Kit of the Invention
[0181] The present invention also provides a kit comprising (i) the
coelenterazine analog of the invention and (ii) at least one
selected from the group consisting of the luciferase, etc., a
polynucleotide encoding the luciferase, etc., a recombinant vector
comprising the polynucleotide, and a transformant comprising the
polynucleotide. A preferred embodiment of the present invention
further provides a kit comprising (i) the coelenterazine analog of
the invention and (ii) at least one selected front the group
consisting of a luciferase, a polynucleotide encoding the
luciferase, a recombinant vector comprising the polynucleotide, and
a transformant comprising the polynucleotide.
[0182] The luciferase used is preferably the 19 kDa protein of
Oplophorus luciferase and its mutated proteins (e.g., the protein
of the present invention), and more preferably, the protein of the
present invention.
[0183] The kit of the invention can be prepared by conventional
methods using conventional materials. The kit of the invention may
additionally comprise, for example, sample tubes, plates,
instructions for the user, solutions, buffers, reagents, and either
samples suitable for standardization or control samples. The kit of
the invention may further comprise salts containing halide
ions.
[0184] The kit of the present invention may be used for the
aforesaid measurement using a reporter protein or a reporter gene,
as a detection marker by luminescence, the analysis of
physiological functions or enzyme activities by the BRET method, or
the like. The kit may also be used for the luminescence method
which will be later described.
8. Method for Luminescence Reaction
Luminescence Reaction
[0185] The luminescence reaction using the coelenterazine analog of
the invention as a substrate can be performed by contacting the
coelenterazine analog of the invention with luciferase. As used
herein, the "contact" is intended to mean that the coelenterazine
analog of the invention and luciferase are allowed to be present in
the same reaction system, and includes, for example, the
coelenterazine analog of the invention being added to a container
charged with a luciferase, the luciferase being added to a
container charged with the coelenterazine analog of the invention,
and the coelenterazine analog of the invention being mixed with the
luciferase. The reaction can be carried out under conditions
conventionally used for the luminescence reaction using Oplophorus
luciferase or under a modification of these conditions.
[0186] Specifically, solvents for the reaction which are employed
are, for example, a buffer solution such as Tris-HCl buffer, sodium
phosphate buffer, etc., water, and the like.
[0187] Temperatures for the reaction are generally at approximately
4.degree. C. to 40.degree. C. and preferably approximately
4.degree. C. to 25.degree. C.
[0188] In the reaction solution, PH is generally approximately 5 to
10, preferably approximately 6 to 9, more preferably approximately
7 to 8 and most preferably approximately 7.5.
[0189] The luciferase used is preferably the 19 kDa protein of
Oplophorus luciferase and its mutated proteins (e.g., the protein
of the present invention), and more preferably, the protein of the
present invention.
[0190] The coelenterazine analog of the present invention may also
be added to the reaction system in the form of a solution in a
polar solvent such as dimethylformamide, dimethylsulfoxide, etc.,
or in an alcohol such as methanol, ethanol, butanol, etc.
Activation of Luminescence Activity
[0191] The luminescence activity of the protein of the invention
wherein the luciferin serves as a substrate is activated by halide
ions, non-ionic surfactants, etc.
[0192] Examples of the halide ions are fluorine ions, chlorine
ions, bromine ions and iodine ions; preferred are chlorine ions,
bromine ions and iodine ions.
[0193] The concentration of the halide ions is generally
approximately 10 .mu.M to 100 mM, preferably approximately 100
.mu.M to 50 mM and particularly preferably approximately 1 mM to 20
nM.
[0194] To add the halide ions to the reaction system, there is a
method which comprises adding these ions in a salt form. The salts
used are alkali metal salts such as sodium salts, potassium salts,
etc.; alkaline earth metal salts such as calcium salts, magnesium
salts, barium salts, etc. More specific examples are NaF, NaCl,
NaBr, NaI, KF, KCl, KBr, KI, CaF.sub.2, CaCl.sub.2, CaBr.sub.2,
CaI.sub.2, MgF.sub.2, MgCl.sub.2, MgBr.sub.2, MgI.sub.2, etc.
[0195] Commercial products (trademark) of the non-ionic surfactants
include Tween 20 (polyoxyethylene sorbitan monolaurate). Tween 80
(polyoxyethylene sorbitan monooleate), Triton X-100 (polyethylene
glycol-p-isooctylphenyl ether), Briji-58 (polyoxyethylene (20)
cetyl ether), Nonidet P-40 (ethylphenol poly(ethylene glycol
ether)n), etc., and preferably, Tween 20, Triton X-100, etc.
[0196] The concentration of the non-ionic surfactants is generally
approximately 0.0002% (w/v) to 0.2% (w/v), preferably approximately
0.001% (w/v) to 0.1%(w/v), and more preferably approximately 0.05%
(w/v) to 0.02%(w/v).
[0197] All literatures and publications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, irrespective of their
purposes.
[0198] Unless otherwise indicated with respect to the embodiments
and working examples, the methods described in standard sets of
protocols such as J. Sambrook, E. F. Fritsch & T. Maniatis
(Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold
Spring Harbor Press, Cold Spring Harbor, New York (2001); F. M.
Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J.
A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology,
John Wiley & Sons Ltd., etc., or modifications or variations
thereof are used. When commercially available reagent kits or
measuring apparatuses are used, protocols attached thereto are used
unless otherwise indicated.
[0199] The objects, characteristics, and advantages of the present
invention as well as the idea thereof are apparent to those skilled
in the art from the descriptions given herein. Based on the
description given herein, those skilled in the art can easily work
the present invention.
[0200] It is to be understood that the mode for carrying out the
invention, specific working examples, etc. are to be taken as
preferred embodiments of the present invention. These descriptions
are only for illustrative and explanatory purposes and are not
intended to restrict the invention thereto. It is further apparent
to those skilled in the art that various modifications may be made
based on the descriptions given herein within the intent and scope
of the present invention disclosed herein.
EXAMPLES
[0201] Hereinafter, the present invention will be described with
reference to EXAMPLES below but is not deemed to limit the
invention thereto.
Example 1
Synthesis of C6d-f-Coelenterazine
1) Synthesis of 1,1-Diethoxy-3-(4-fluoropohenyl)propane-2-one
##STR00011##
[0203] Under argon atmosphere, a diethyl ether solution of
(4-fluorobenzyl)magnesium chloride (0.25 M, 20.0 mL, 5.0 mmol) was
added slowly to a solution of ethyl diethoxyacetate (890 .mu.L,
5.00 mmol) in tetrahydrofuran (THF) (50 mL) at -78.degree. C. After
stirring at -78.degree. C. for 2 hours, the temperature was
gradually elevated to room temperature, followed by stirring for 12
hours. To the mixture was added aqueous 20% ammonium chloride
solution (10 mL). The mixture was extracted with ethyl acetate
(.times.3). The organic layer was successively washed with water
(.times.1) and brine (.times.1) and then dried over anhydrous
sodium sulfate. After filtration, the filtrate was concentrated
under reduced pressure and the residue was purified by silica gel
flash column chromatography (n-hexane/ethyl
acetate=19/1.fwdarw.4/1) to give
1,1-diethoxy-3-(4-fluorophenyl)propan-2-one as a colorless oily
substance (622 mg, 2.59 mmol, 51.7%).
[0204] TLC R.sub.f=0.24 (n-hexane/ethyl acetate=20/1);
[0205] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 1.25 (t, 6H, J=7.0
Hz), 3.55 (dq, 2H, J=7.5, 9.5 Hz), 3.68 (dq, 2H, J=7.5, 9.5 Hz),
3.86 (s, 2H), 4.62 (s, 1H), 6.97-7.02 (m, 2H), 7.15-7.19 (m,
2H),
2) Synthesis of C6d-f-Coelenterazine (C6d-f-CTZ)
##STR00012##
[0207] Under argon atmosphere,
1,1-diethoxy-3-(4-fluorophenyl)propan-2-one (333 mg, 1.39 mmol) was
dissolved in 1,4-dioxane (2.0 mL) and water (0.4 mL). To the
solution was added 2-amino-3-benzyl-5-phenylpyrazine (261 mg, 1.00
mmol). After cooling to 0.degree. C., cone. hydrochloric acid (0.20
mL) was added to the mixture, which was stirred at 100.degree. C.
for overnight (12 hours), The mixture was allowed to cool to room
temperature and concentrated under reduced pressure. The residue
was purified in an argon flow by silica gel flash column
chromatography (n-hexane/ethyl acetate=5/1.fwdarw.1/1.fwdarw.ethyl
acetate.fwdarw.ethyl acetate/methanol=50/1.fwdarw.20/1) to give the
crude product (264 mg, <0.645 mmol) containing C6d-f-CTZ as a
major compound. The resulting solid was further purified by
reprecipitation (n-hexane/methanol) to give C6d-f-CTZ as a pale
brown powder (49.2 mg, 120 .mu.mol, 12.0%).
[0208] TLC R.sub.f=0.35 (ethyl acetate);
[0209] .sup.1H NMR (500 MHz, CD.sub.3OD) .delta. 4.29 (s, 2H), 4.58
(s, 2H), 7.05-7.09 (m, 2H), 7.24-7.28 (m, 1H), 7.31-7.34 (m, 4H),
7.42 (d, 2H, J=7.0 Hz), 7.49-7.56 (m, 3H), 7.93 (br d, 2H, J=7.0
Hz) 8.54 (s, 1H),
Example 2
Desion and Chemical Synthesis of Codon-Optimized Nucleic Acid
[0210] Based on the amino acid sequence (SEQ ID NO: 1) of the
catalytic 19 kDa domain (sometimes referred to as "nanoLuc") of the
mutated Oplophorus luciferase from Promega Corp., which was
generated by mutagenesis of the catalytic 19 kDa domain (sometimes
referred to as "KAZ") of native Oplophorus luciferase, a
codon-optimized gene for the mutated catalytic 19 kDa domain
(sometimes referred to as "nanoKAZ") of Oplophorus luciferase was
designed. More specifically, the nucleotide sequence (SEQ ID NO: 2)
of nanoKAZ was designed, without changing the amino acid sequence
(SEQ ID NO: 1) of nanoLuc, using only the amino acid codon
frequently used in human. The codon-optimized gene for the nanoKAZ
domain was chemically synthesized in a conventional manner.
Example 3
Construction of Vector for Secretion and Expression of
Condon-Optimized nanoKAZ Domain Protein Using Secretory Signal
Sequence of Gaussia Luciferase in Cultured Animal Cells
[0211] A vector for expression of the codon-optimized nanoKAZ
domain protein was constructed as follows.
[0212] Firstly, a novel expression vector pcDNA3-GLsp in cultured
animal cells was constructed (FIG. 1). Specifically, the secretory
signal sequence of Gaussia luciferase was obtained from the
pcDNA3-GLuc vector (manufactured by Prolume Ltd.) by PCR using the
primer set of GLsp-1R/EcoRI (SEQ ID NO: 3: 5' ggc GAA TTC GGT GGG
CTT GGC CTC GGC CAC 3', EcoRI sequence underlined) and T7 primer
(SEQ ID NO: 4: 5' TAATACG ACTCACTATAGGG 3') and digested with
HindIII/EcoRI. The resulting fragment was inserted into the
HindIII/EcoRI sites which are the restriction enzyme site of the
pcDNA3 vector (manufactured by Invitrogen Corp.) to construct the
novel expression vector of pcDNA3-GLsp, That is, the novel
expression vector is under the control of the CMV promoter and has
a Kozak sequence, followed by the secretory signal sequence of
Gaussia luciferase and the multiple cloning site sequence.
[0213] Next, the codon-optimized nanoKAZ domain protein expression
vector pcDNA3-GLsp-nanoKAZ (FIG. 2) was constructed as follows,
using the novel expression vector pcDNA3-GLsp.
[0214] The gene fragment of the codon-optimized nanoKAZ domain was
digested with the restriction enzyme of EcoRI/XbaI in a
conventional manner and then was ligated to the EcoRI-XbaI site of
pcDNA3-GLsp to construct the expression vector pcDNA3-GLsp-nanoKAZ,
as shown in FIG. 2. The sequence of the inserted gene was confirmed
by DNA sequencing using a DNA Sequencer (manufactured by ABI
Co.).
[0215] The nucleotide sequence of GLsp-nanoKAZ encoded in the
expression vector pcDNA3-GLsp-nanoKAZ is shown by SEQ ID NO: 5 and
the amino acid sequence is shown by SEQ ID NO: 6. The amino acid
sequence after secretion is shown by SEQ ID NO: 7.
Example 4
Transfection of Vector into Cultured Animal Cells and Preparation
of Enzyme Used for Assay
(1) Purification of Expression Plasmid
[0216] Using the pcDNA3-GLsp-nanoKAZ plasmid obtained in EXAMPLE 3,
the following experiment was performed. The pcDNA3-GLsp-nanoKAZ
plasmid was purified in Escherichia coli JM83 strain by using a
Plasmid Purification Kit (manufactured by QIAGEN, Inc.), and
dissolved in sterile water to a concentration of 1 .mu.g/.mu.L.
(2) Transfection and Preparation of Enzyme for Assay
[0217] The Chinese hamster ovary-derived cell line CHO-KI was
cultured in Ham's F-12 medium (manufactured by Wako Pure Chemical
Industries, Ltd.) containing 10% (v/v) calf fetal serum
(manufactured by Biowest Inc.), and cultured in DMEM (manufactured
by Wako Pure Chemical Industries, Ltd.) containing 10% (v/v) calf
fetal serum (manufactured by Biowest Inc.). The respective cells
were seeded in a 6-well culture plate (n=2) with 1.times.10.sup.5
cells/well/2 mL medium, and cultured in an incubator at 37.degree.
C. in 5% CO.sub.2, respectively. After 24 hours, the purified
pcDNA3-GLsp-nanoKAZ plasmid was transfected into CHO-K1 cells using
a FuGene HD Transfection Kit (manufactured by Promega Corp.), which
was used in the subsequent experiment. Specifically, 1 .mu.g of the
pcDNA3-GLsp-nanoKAZ expression vector and 3 .mu.L of FuGene HD were
added to 100 .mu.L of medium, which was allowed to stand at room
temperature for 15 minutes. Then 100 .mu.L of the DNA-FuGene
complex solution was added to the cells in the 6 wells. After
incubation for 48 hours, the culture medium was recovered and used
as the secreted nanoKAZ enzyme solution for assay.
Example 5
Assay for Luminescence Activity in Codon-Optimized nanoKAZ Domain
Protein Expressed in Cultured Animal Cells
[0218] The enzyme solution, 5 .mu.L, for assay obtained in EXAMPLE
4 (2) was added to 100 .mu.L of 50 mM Tris-HCl (pH 7.6)-10 mM EDTA
(Wako Pure Chemical Industries) containing 1 .mu.g each of various
coelenterazine analogs to initiate the luminescence reaction. The
luminescence activity was determined by a luminometer (AB2200,
manufactured by Atto Company) for 60 seconds and expressed by the
maximum luminescence intensity (I.sub.max) and the relative light
units (rlu) of the lumimescence intenisity integrated for 60
seconds.
Example 6
Substrate Specificity of Condon-Optimized nanoKAZ Domain Protein
and Comparison of Luminescence Patterns
[0219] The coelenterazine analogs used for substrate specificity
analysis except for the analog described in EXAMPLE 1 were
synthesized by the methods as described in the journals below,
respectively. Specifically, bis-coelenterazine was synthesized by
the method described in Nakamura et al. (1997) Tetrahedron Lett.
38: 6405-6406; Furimazine (Compound No. 3939 referred to in the
journal) and C6d-coelenterazine (Compound No. 3840 referred to in
the journal) by the method described in Hall et al. (2012) ACS
Chem. Biol. 16: 848-1857; and the C2-coelenterazine analogs by the
method described in Inouye et al (2010) Anal. Biochem. 407:
247-252. The enzyme solution of the culture medium in which nanoKAZ
was secreted by the method as described in EXAMPLE 4 (2) was used
for determining the luminescence activity with coelenterazine or
its analogs.. As a result, the substrate specificities for nanoKAZ
are summarized in TABLE 1. In the coelenterazine analogs, the
compounds exhibiting the relative maximum luminescence intensity 5
times or higher than that of coelenterazine were h-, bis-, f-, and
C6d-f-coelenterazines and furimazine, and the compounds exhibiting
the relative amount of the integrated luminescence intensity for 1
min with 5 times or higher than that of coelenterazine were h-, f-,
and C6d-f-coelenterazines. On the other hand, the luminescence
patterns for these compounds are shown in FIG. 3. As shown in FIG.
3, his-coelenterazine, furimazine and C6d-ficoelenterazine did not
exhibit a rapid decay of the luminescence patterns. As a result,
the luminescence substrates which can markedly improve the
luminescence intensity and the amount of the integrated
luminescence intensity to compare with furimazine are
bis-coelenterazine and C6d-f-coelenterazine, showing a continuous
luminescence without a decay of the luminescence intensity.
TABLE-US-00001 TABLE 1 Substrate Specificity of Codon-Optimized
nanoKAZ Domain Protein Relative amount of integrated Relative
maximum luminescence Coelenterazine analog luminescence intensity
(I.sub.max) intensity (1 min.) Coelenterazine (CTZ) 1.0 1.0
C6d-Coelenterazine 0.8 0.7 h-Coelenterazine 17.0 10.1
bis-Coelenterazine 10.3 9.7 f-Coelenterazine 19.5 11.1
C6d-f-Coelenterazine 10.1 10.1 Furimazine 5.5 5.7
Example 7
Substrate Specificity of Various Luciferases
[0220] The preparation of various luciferases and the determination
of their luminescence activities were carried out in accordance
with the methods as previously reported. The catalytic 19 kDa
domain of Oplophorus luciferase was produced by the method
described in Inouye S. & Sasaki S. (2007) Protein Express.
Purif. 56, 261-268, Gaussia luciferase by the method described in
Inouye S & Sahara Y. (2008) Biochem, Biophys. Res. Commun, 368,
600-605, Renilla luciferase and Renilla luciferase 547 by the
method described in Inouye S. et al. (2013) Protein Express. Purif.
88, 150-156, and aequorin by the method described in Inouye S et
al. (2010) Anal. Biochem, 407, 247-252, respectively, and used. The
results of assay are shown in TABLE 2. Especially, novel
C6d-f-coelenterazine was found to be a good substrate for the
catalytic 19 kDa domain of Oplophorus luciferase.
TABLE-US-00002 TABLE 2 Substrate Specificities for
Coelenterazine-type Luciferases and Photoprotein Maximum
Luminescence Intensity (I.sub.max, %) (Relative Amount of
Integrated Luminescence intensity (1 min, %)) Catalytic 19 kDa
domain of Oplophorus Gaussia Renilla Renilla Coelenterazine analog
luciferase luciferase luciferase luciferase 547 Aequorin
Coelenterazine (CTZ) 100 (100) 100 (100) 100 (100) 100 (100) 100
(100) C6d-Coelenterazine 9.0 (7.7) 0.8 (0.8) 0.02 (0.02) 0.12
(0.12) 0.03 (0.03) h-Coelenterazine 92.6 (101) 8.2 (12.1) 68.5
(73.5) 24.5 (16.4) 84.0 (68.0) bis-Coelenterazine 108 (102) 0 (0)
0.04 (0.03) 0.07 (0.08) 0.01 (0.01) f-Coelenterazine 78.6 (85.7)
5.0 (10) 65.3 (71.4) 78.0 (68.1) 42.3 (59.0) C6d-f-Coelenterazine
100 (105) 0 (0) 0.07 (0.01) 0.17 (0.13) 0.12 (0.19) Furimazine 72.2
(652) 0 (0) 0.01 (0.01) 0.01 (0.01) 0.02 (0.02)
Example 8
Evaluation of Stability of Coelenterazine Analogs in Aqueous
Solution
[0221] The stability of coelenterazine analogs in an aqueous
solution was examined by dissolving 10 .mu.g of coelenterazine
analogs in 0.5 mL of 30 mM Tris-HCl (pH 7.6), allowing the solution
to stand at 37.degree. C. for 22 hours, and then the degradation of
coelenterazine analogs was determined by HPLC analysis. The HPLC
analysis was performed by the modified method as described in
Inouye S. et al. (2013) Protein Express. Purif 88, 150-156.
[0222] The results are summarized in TABLE 3. After incubation for
22 hours, coelenterazine and furimazine were very unstable, and
only 2% and 3% of undegraded compounds remained, respectively. On
the other hand, 38% and 49% of bis-coelenterazine and
C6d-f-coelenterazine remained, respectively. That is, it was found
that C6d-f-coelenterazine was the most stable coelenterazine
analog, indicating that the stability was markedly improved, by
comparison to native coelenterazine.
TABLE-US-00003 TABLE 3 Comparison in Stability of Coelenterazine
Analog in Aqueous Solution Coelenterazine Incubation Time at
Residual Ratio of Analog 37.degree. C. (hour) Coelenterazine Analog
(%) Coelenterazine 0 100 22 2 bis-Coelenterazine 0 100 22 38
C6d-f-Coelenterazine 0 100 22 49 Furimazine 0 100 22 3
[Sequence Listing Free Text]
[SEQ ID NO: 1]
[0223] Amino acid sequence of nanoLuc and nanoKAZ.
[SEQ II) NO: 2]
[0224] Nucleotide sequence of the polynucleotide encoding
nanoKAZ.
[SEQ ID NO: 3]
[0225] Nucleotide sequence of the primer used in EXAMPLE 3.
[SEQ ID NO: 4]
[0226] Nucleotide sequence of the primer used in EXAMPLE 3.
[SEQ ID NO: 5]
[0227] Nucleotide sequence of the polynucleotide encoding
GLsp-nanoKAZ.
[SEQ ID NO: 6]
[0228] Amino acid sequence of GLsp-nanoKAZ.
[SEQ ID NO: 7]
[0229] Amino acid sequence of the secreted nanoKAZ
[SEQ ID NO: 8]
[0230] Nucleotide sequence encoding the 19 kDa protein of
Oplophorus gracilorostris luciferase having the catalytic function
of luminescence.
[SEQ II) NO: 9]
[0231] Amino acid sequence of the 19 kDa protein of Oplophorus
gracilorostris luciferase having the catalytic function of
luminescence.
Sequence CWU 1
1
91171PRTArtificial sequenceSynthetic Construct 1Met Val Phe Thr Leu
Glu Asp Phe Val Gly Asp Trp Arg Gln Thr Ala 1 5 10 15 Gly Tyr Asn
Leu Asp Gln Val Leu Glu Gln Gly Gly Val Ser Ser Leu 20 25 30 Phe
Gln Asn Leu Gly Val Ser Val Thr Pro Ile Gln Arg Ile Val Leu 35 40
45 Ser Gly Glu Asn Gly Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr
50 55 60 Glu Gly Leu Ser Gly Asp Gln Met Gly Gln Ile Glu Lys Ile
Phe Lys 65 70 75 80 Val Val Tyr Pro Val Asp Asp His His Phe Lys Val
Ile Leu His Tyr 85 90 95 Gly Thr Leu Val Ile Asp Gly Val Thr Pro
Asn Met Ile Asp Tyr Phe 100 105 110 Gly Arg Pro Tyr Glu Gly Ile Ala
Val Phe Asp Gly Lys Lys Ile Thr 115 120 125 Val Thr Gly Thr Leu Trp
Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu 130 135 140 Ile Asn Pro Asp
Gly Ser Leu Leu Phe Arg Val Thr Ile Asn Gly Val 145 150 155 160 Thr
Gly Trp Arg Leu Cys Glu Arg Ile Leu Ala 165 170 2516DNAArtificial
sequencesynthetic DNA 2atggtcttca ccctggagga cttcgtcggc gactggagac
agaccgccgg ctacaacctg 60gaccaggtcc tggagcaggg cggcgtcagc agcctgttcc
agaacctggg cgtcagcgtc 120acccccatcc agagaatcgt ccttagcggc
gagaacggcc tgaagatcga catccacgtc 180atcatcccct acgagggcct
gagcggcgac cagatgggcc agatcgagaa gatcttcaag 240gtcgtctacc
ccgtcgacga ccaccacttc aaggtcatcc tgcactacgg caccctggtc
300atcgacggcg tcacccccaa catgatcgac tacttcggta gaccctacga
gggcatcgcc 360gtcttcgacg gcaagaagat caccgtcacc ggcaccctgt
ggaacggcaa caagatcatc 420gacgagagac tgatcaaccc cgacggcagc
ctgctgttca gagtcaccat caacggcgtc 480accggctgga gactgtgcga
gagaatcctg gcctaa 516330DNAArtificial sequencesynthetic DNA
3ggcgaattcg gtgggcttgg cctcggccac 30420DNAArtificial
sequencesynthetic DNA 4taatacgact cactataggg 205597DNAArtificial
sequencesynthetic DNA 5atg gga gtc aaa gtt ctg ttt gcc ctg atc tgc
atc gct gtg gcc gag 48Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys
Ile Ala Val Ala Glu 1 5 10 15 gcc aag ccc acc gaa ttc aag ctt ggt
acc acc atg gtc ttc acc ctg 96Ala Lys Pro Thr Glu Phe Lys Leu Gly
Thr Thr Met Val Phe Thr Leu 20 25 30 gag gac ttc gtc ggc gac tgg
aga cag acc gcc ggc tac aac ctg gac 144Glu Asp Phe Val Gly Asp Trp
Arg Gln Thr Ala Gly Tyr Asn Leu Asp 35 40 45 cag gtc ctg gag cag
ggc ggc gtc agc agc ctg ttc cag aac ctg ggc 192Gln Val Leu Glu Gln
Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly 50 55 60 gtc agc gtc
acc ccc atc cag aga atc gtc ctt agc ggc gag aac ggc 240Val Ser Val
Thr Pro Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly 65 70 75 80 ctg
aag atc gac atc cac gtc atc atc ccc tac gag ggc ctg agc ggc 288Leu
Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Gly 85 90
95 gac cag atg ggc cag atc gag aag atc ttc aag gtc gtc tac ccc gtc
336Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val Tyr Pro Val
100 105 110 gac gac cac cac ttc aag gtc atc ctg cac tac ggc acc ctg
gtc atc 384Asp Asp His His Phe Lys Val Ile Leu His Tyr Gly Thr Leu
Val Ile 115 120 125 gac ggc gtc acc ccc aac atg atc gac tac ttc ggt
aga ccc tac gag 432Asp Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly
Arg Pro Tyr Glu 130 135 140 ggc atc gcc gtc ttc gac ggc aag aag atc
acc gtc acc ggc acc ctg 480Gly Ile Ala Val Phe Asp Gly Lys Lys Ile
Thr Val Thr Gly Thr Leu 145 150 155 160 tgg aac ggc aac aag atc atc
gac gag aga ctg atc aac ccc gac ggc 528Trp Asn Gly Asn Lys Ile Ile
Asp Glu Arg Leu Ile Asn Pro Asp Gly 165 170 175 agc ctg ctg ttc aga
gtc acc atc aac ggc gtc acc ggc tgg aga ctg 576Ser Leu Leu Phe Arg
Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu 180 185 190 tgc gag aga
atc ctg gcc taa 597Cys Glu Arg Ile Leu Ala 195 6198PRTArtificial
sequenceSynthetic Construct 6Met Gly Val Lys Val Leu Phe Ala Leu
Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Lys Pro Thr Glu Phe Lys
Leu Gly Thr Thr Met Val Phe Thr Leu 20 25 30 Glu Asp Phe Val Gly
Asp Trp Arg Gln Thr Ala Gly Tyr Asn Leu Asp 35 40 45 Gln Val Leu
Glu Gln Gly Gly Val Ser Ser Leu Phe Gln Asn Leu Gly 50 55 60 Val
Ser Val Thr Pro Ile Gln Arg Ile Val Leu Ser Gly Glu Asn Gly 65 70
75 80 Leu Lys Ile Asp Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser
Gly 85 90 95 Asp Gln Met Gly Gln Ile Glu Lys Ile Phe Lys Val Val
Tyr Pro Val 100 105 110 Asp Asp His His Phe Lys Val Ile Leu His Tyr
Gly Thr Leu Val Ile 115 120 125 Asp Gly Val Thr Pro Asn Met Ile Asp
Tyr Phe Gly Arg Pro Tyr Glu 130 135 140 Gly Ile Ala Val Phe Asp Gly
Lys Lys Ile Thr Val Thr Gly Thr Leu 145 150 155 160 Trp Asn Gly Asn
Lys Ile Ile Asp Glu Arg Leu Ile Asn Pro Asp Gly 165 170 175 Ser Leu
Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu 180 185 190
Cys Glu Arg Ile Leu Ala 195 7181PRTArtificial sequenceSynthetic
Construct 7Lys Pro Thr Glu Phe Lys Leu Gly Thr Thr Met Val Phe Thr
Leu Glu 1 5 10 15 Asp Phe Val Gly Asp Trp Arg Gln Thr Ala Gly Tyr
Asn Leu Asp Gln 20 25 30 Val Leu Glu Gln Gly Gly Val Ser Ser Leu
Phe Gln Asn Leu Gly Val 35 40 45 Ser Val Thr Pro Ile Gln Arg Ile
Val Leu Ser Gly Glu Asn Gly Leu 50 55 60 Lys Ile Asp Ile His Val
Ile Ile Pro Tyr Glu Gly Leu Ser Gly Asp 65 70 75 80 Gln Met Gly Gln
Ile Glu Lys Ile Phe Lys Val Val Tyr Pro Val Asp 85 90 95 Asp His
His Phe Lys Val Ile Leu His Tyr Gly Thr Leu Val Ile Asp 100 105 110
Gly Val Thr Pro Asn Met Ile Asp Tyr Phe Gly Arg Pro Tyr Glu Gly 115
120 125 Ile Ala Val Phe Asp Gly Lys Lys Ile Thr Val Thr Gly Thr Leu
Trp 130 135 140 Asn Gly Asn Lys Ile Ile Asp Glu Arg Leu Ile Asn Pro
Asp Gly Ser 145 150 155 160 Leu Leu Phe Arg Val Thr Ile Asn Gly Val
Thr Gly Trp Arg Leu Cys 165 170 175 Glu Arg Ile Leu Ala 180
8591DNAOplophorus gracilorostrisCDS(1)..(591) 8atg gcg tac tcc act
ctg ttc ata att gca ttg acc gcc gtt gtc act 48Met Ala Tyr Ser Thr
Leu Phe Ile Ile Ala Leu Thr Ala Val Val Thr 1 5 10 15 caa gct tcc
tca act caa aaa tct aac cta act ttt acg ttg gca gat 96Gln Ala Ser
Ser Thr Gln Lys Ser Asn Leu Thr Phe Thr Leu Ala Asp 20 25 30 ttc
gtt gga gac tgg caa cag aca gct gga tac aac caa gat caa gtg 144Phe
Val Gly Asp Trp Gln Gln Thr Ala Gly Tyr Asn Gln Asp Gln Val 35 40
45 tta gaa caa gga gga ttg tct agt ctg ttc caa gcc ctg gga gtg tca
192Leu Glu Gln Gly Gly Leu Ser Ser Leu Phe Gln Ala Leu Gly Val Ser
50 55 60 gtc acg ccc ata cag aaa gtt gta ctg tct ggg gag aat ggg
tta aaa 240Val Thr Pro Ile Gln Lys Val Val Leu Ser Gly Glu Asn Gly
Leu Lys 65 70 75 80 gct gat att cat gtc ata ata cct tac gag gga ctc
agt ggt ttt caa 288Ala Asp Ile His Val Ile Ile Pro Tyr Glu Gly Leu
Ser Gly Phe Gln 85 90 95 atg ggt cta att gaa atg atc ttc aaa gtt
gtt tac ccc gtg gat gat 336Met Gly Leu Ile Glu Met Ile Phe Lys Val
Val Tyr Pro Val Asp Asp 100 105 110 cat cat ttc aag att att ctc cat
tat ggt aca ctc gtt att gac ggt 384His His Phe Lys Ile Ile Leu His
Tyr Gly Thr Leu Val Ile Asp Gly 115 120 125 gta aca ccc aac atg att
gac tac ttt gga aga cct tac cct gga att 432Val Thr Pro Asn Met Ile
Asp Tyr Phe Gly Arg Pro Tyr Pro Gly Ile 130 135 140 gct gta ttt gac
ggc aag cag atc aca gtt act gga act ctg tgg aac 480Ala Val Phe Asp
Gly Lys Gln Ile Thr Val Thr Gly Thr Leu Trp Asn 145 150 155 160 ggc
aac aag atc tat gat gag agg cta atc aac cct gat ggt tca ctc 528Gly
Asn Lys Ile Tyr Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu 165 170
175 ctc ttc aga gtt act atc aat gga gtc acg gga tgg agg ctt tgc gag
576Leu Phe Arg Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu
180 185 190 aac att ctt gcc taa 591Asn Ile Leu Ala 195
9196PRTOplophorus gracilorostris 9Met Ala Tyr Ser Thr Leu Phe Ile
Ile Ala Leu Thr Ala Val Val Thr 1 5 10 15 Gln Ala Ser Ser Thr Gln
Lys Ser Asn Leu Thr Phe Thr Leu Ala Asp 20 25 30 Phe Val Gly Asp
Trp Gln Gln Thr Ala Gly Tyr Asn Gln Asp Gln Val 35 40 45 Leu Glu
Gln Gly Gly Leu Ser Ser Leu Phe Gln Ala Leu Gly Val Ser 50 55 60
Val Thr Pro Ile Gln Lys Val Val Leu Ser Gly Glu Asn Gly Leu Lys 65
70 75 80 Ala Asp Ile His Val Ile Ile Pro Tyr Glu Gly Leu Ser Gly
Phe Gln 85 90 95 Met Gly Leu Ile Glu Met Ile Phe Lys Val Val Tyr
Pro Val Asp Asp 100 105 110 His His Phe Lys Ile Ile Leu His Tyr Gly
Thr Leu Val Ile Asp Gly 115 120 125 Val Thr Pro Asn Met Ile Asp Tyr
Phe Gly Arg Pro Tyr Pro Gly Ile 130 135 140 Ala Val Phe Asp Gly Lys
Gln Ile Thr Val Thr Gly Thr Leu Trp Asn 145 150 155 160 Gly Asn Lys
Ile Tyr Asp Glu Arg Leu Ile Asn Pro Asp Gly Ser Leu 165 170 175 Leu
Phe Arg Val Thr Ile Asn Gly Val Thr Gly Trp Arg Leu Cys Glu 180 185
190 Asn Ile Leu Ala 195
* * * * *