U.S. patent application number 17/202064 was filed with the patent office on 2022-02-03 for identification of channelrhodopsin-2 (chr2) mutations and methods of use.
The applicant listed for this patent is Wayne State University. Invention is credited to Zhuo-Hua PAN.
Application Number | 20220033449 17/202064 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-03 |
United States Patent
Application |
20220033449 |
Kind Code |
A1 |
PAN; Zhuo-Hua |
February 3, 2022 |
IDENTIFICATION OF CHANNELRHODOPSIN-2 (CHR2) MUTATIONS AND METHODS
OF USE
Abstract
The invention provides compositions and kits including at least
one nucleic acid or polypeptide molecule encoding for a mutant ChR2
protein. Methods of the invention include administering a
composition comprising a mutant ChR2 to a subject to preserve,
improve, or restore phototransduction. Preferably, the compositions
and methods of the invention are provided to a subject having
impaired vision, thereby restoring vision to normal levels.
Inventors: |
PAN; Zhuo-Hua; (Troy,
MI) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Wayne State University |
Detroit |
MI |
US |
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Appl. No.: |
17/202064 |
Filed: |
March 15, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14383211 |
Sep 5, 2014 |
10947281 |
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PCT/US13/29171 |
Mar 5, 2013 |
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17202064 |
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61606663 |
Mar 5, 2012 |
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International
Class: |
C07K 14/405 20060101
C07K014/405; A61K 38/16 20060101 A61K038/16; C07K 14/00 20060101
C07K014/00; A61P 27/02 20060101 A61P027/02 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with U.S. Government support under
the National Institutes of Health/National Eye Institute grant NIH
EY 17130. The Government has certain rights in the invention.
Claims
1.-50. (canceled)
51. An expression vector comprising a polynucleotide encoding a
polypeptide comprising the amino acid sequence of SEQ ID NO: 26 in
which the amino acid at position 132 of SEQ ID NO: 26 is cysteine
(C) or alanine (A), and the amino acid at position 159 of SEQ ID
NO: 26 is cysteine (C), serine (S), or alanine (A).
52. The expression vector of claim 51, wherein the polypeptide
comprises a cysteine (C) at positions 132 and 159 of SEQ ID NO:
26.
53. The expression vector of claim 52, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO: 16 and the
polynucleotide comprises the nucleic acid sequence of SEQ ID NO:
15.
54. The expression vector of claim 51, wherein the polypeptide
comprises a cysteine (C) at position 132 and a serine (S) at
position 159 of SEQ ID NO: 26.
55. The expression vector of claim 54, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO: 19 and the
polynucleotide comprises the nucleic acid sequence of SEQ ID NO:
18.
56. The expression vector of claim 51, wherein the polypeptide
comprises a cysteine (C) at position 132 and an alanine (A) at
position 159 of SEQ ID NO: 26.
57. The expression vector of claim 56, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO: 25 and the
polynucleotide comprises the nucleic acid sequence of SEQ ID NO:
24.
58. The expression vector of claim 51, wherein the polypeptide
comprises an alanine (A) at position 132 and a cysteine (C) at
position 159 of SEQ ID NO: 26.
59. The expression vector of claim 58, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO: 22 and the
polynucleotide comprises the nucleic acid sequence of SEQ ID NO:
21.
60. The expression vector of claim 51, wherein the polypeptide
comprises an alanine (A) at position 132 and a serine (S) at
position 159 of SEQ ID NO: 26.
61. The expression vector of claim 51, wherein the polypeptide
comprises an alanine (A) at position 132 and an alanine (A) at
position 159 of SEQ ID NO: 26.
62. The expression vector of claim 51, wherein the expression
vector is an adeno-associated virus (AAV) vector.
63. The expression vector of claim 62, wherein the AAV vector is an
AAV2 vector.
64. A method of improving or restoring vision in a subject, the
method comprising administering to the subject the expression
vector of claim 51.
65. A method of improving or restoring vision in a subject, the
method comprising administering to the subject the expression
vector of claim 62.
66. A method of improving or restoring vision in a subject, the
method comprising administering to the subject the expression
vector of claim 63.
67. The method of claim 64, wherein the subject either has normal
vision or the subject is suffering from an ocular disease.
68. The method of claim 65, wherein the subject either has normal
vision or the subject is suffering from an ocular disease.
69. The method of claim 66, wherein the subject either has normal
vision or the subject is suffering from an ocular disease.
70. The method of claim 67, wherein the subject is suffering from
an ocular disease and the ocular disease is macular degeneration or
retinitis pigmentosa.
71. The method of claim 68, wherein the subject is suffering from
an ocular disease and the ocular disease is macular degeneration or
retinitis pigmentosa.
72. The method of claim 69, wherein the subject is suffering from
an ocular disease and the ocular disease is macular degeneration or
retinitis pigmentosa.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 14/383,211, filed Sep. 5, 2014, now
U.S. Pat. No. 10,947,281, issued Mar. 16, 2021, which is a national
stage application filed under 35 U.S.C. .sctn. 371, of
International Application No. PCT/US2013/029171, filed on Mar. 5,
2013, which claims the benefit of and priority under 35 U.S.C.
.sctn. 119(e) to U.S. Provisional Application No. 61/606,663, filed
on Mar. 5, 2012, the contents of each of which are incorporated by
reference herein in their entireties.
DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY
[0003] The contents of the text file submitted electronically
herewith are incorporated herein by reference in their entirety: A
computer readable format copy of the Sequence Listing (filename:
052522_506C01US_SL25.txt, date recorded: Mar. 15, 2021, file size
98.2 kilobytes).
FIELD OF THE INVENTION
[0004] This invention relates generally to the field of molecular
biology. Mutations in the Channelopsin-2 (Chop2) gene are
identified. Compositions comprising a mutant Chop2 gene are used in
therapeutic methods to improve and restore vision loss.
BACKGROUND OF THE INVENTION
[0005] The retina is composed of photoreceptors (or photoreceptor
cells, rods and cones). Photoreceptors are highly specialized
neurons that are responsible for phototransduction, or the
conversion of light (in the form of electromagnetic radiation) into
electrical and chemical signals that propagate a cascade of events
within the visual system, ultimately generating a representation of
our world.
[0006] Photoreceptor loss or degeneration severely compromises, if
not completely inhibits, phototransduction of visual information
within the retina. Loss of photoreceptor cells and/or loss of a
photoreceptor cell function are the primary causes of diminished
visual acuity, diminished light sensitivity, and blindness. There
is a long-felt need in the art for compositions and method that
restore photosensitivity of the retina of a subject experiencing
vision loss.
SUMMARY OF THE INVENTION
[0007] The invention provides a solution for the long-felt need for
a method of restoring and/or increasing the light sensitivity of
photoreceptor cells by expression of advantageous mutations, and/or
combinations thereof, of the Channelopsin-2 (Chop2) gene, and
subsequently providing methods for Channelopsin-2 (Chop2)-based
gene therapy.
[0008] Channelopsin-2 (Chop2)-based gene therapy offers a superior
strategy for restoring retinal photosensitivity after photoreceptor
degeneration. The protein product of the Chop2 gene, when bound to
the light-isomerizable chromophore all-trans-retinal, forms a
functional light-gated channel, called channelrhodopsin-2 (ChR2).
Native ChR2 shows low light sensitivity. Recently, two mutant
ChR2s, L132C and T159C, were reported to markedly increase their
light sensitivity (Kleinlogel et al. (2011) Nat Neurosci. 14:513-8;
Berndt et al. (2011) Proc Natl Acad Sci USA. 108:7595-600; Prigge
et al. (2012) J Biol Chem. 287(38)3104:12; the contents of each of
which are incorporated herein in their entireties). The properties
of these two ChR2 mutants (i.e., L132C and T159C) were examined and
compared with a number of double mutants at these two sites to
identify suitable candidates for therapeutic methods. Compositions
comprising one or more of these mutations are provided to a subject
in need thereof for the purpose of restoring vision. Specifically,
desired mutations in the Chop2 gene are introduced to a cell and/or
integrated into the genomic DNA of a cell to improve or restore
vision. Desired mutations in the Chop2 gene that are introduced to
a cell to improve or restore vision may also remain episomal, not
having integrated into the genomic DNA.
[0009] Mutations at the L132 or T159 amino acid positions of Chop2
(and therefore, the resulting ChR2) markedly lower the threshold
light intensity that is required to elicit the ChR2-mediated
photocurrent. Double mutants at the amino acid positions L132 and
T159 further increase the photocurrent at low light intensities,
exceeding that of either of the corresponding single mutations.
Retinal ganglion cells expressing the double mutants at the L132
and T159 positions can respond to light intensities that fall
within the range of normal outdoor lighting conditions but should
still maintain adequate, and high temporal resolution that are
suitable for restoring useful vision. Thus, mutant Chop2 protein of
the present invention that form mutant ChR2s having improved light
sensitivity are used alone or in combination to restore or improve
vision.
[0010] Specifically, the invention provides an isolated polypeptide
molecule comprising or consisting of SEQ ID NO: 26 in which the
amino acid at position 132 of SEQ ID NO: 26 is not leucine (L). In
certain embodiments of the isolated polypeptide molecule, the amino
acid at position 132 is cysteine (C) or alanine (A). When the amino
acid at position 132 is cysteine (C), the polypeptide molecule may
comprise or consist of SEQ ID NO: 13. When the amino acid at
position 132 is alanine (A), the polypeptide molecule may comprise
or consist of SEQ ID NO: 20.
[0011] The invention provides an isolated polypeptide molecule
comprising or consisting of SEQ ID NO: 26 in which the amino acid
at position 159 of SEQ ID NO: 26 is not a threonine (T). In certain
embodiments of the isolated polypeptide molecule, the amino acid at
position 159 is cysteine (C), serine (S), or alanine (A). When the
amino acid at position 159 is cysteine (C), the polypeptide
molecule may comprise or consist of SEQ ID NO: 14. When the amino
acid at position 159 is serine (S), the polypeptide molecule may
comprise or consist of SEQ ID NO: 17. When the amino acid at
position 159 is alanine (A), the polypeptide molecule may comprise
or consist of SEQ ID NO: 23.
[0012] The invention provides isolated polypeptide molecule
comprising or consisting of SEQ ID NO: 26 in which the amino acid
at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino
acid at position 159 is not threonine (T). In certain embodiments
of the isolated polypeptide molecule comprising or consisting of
SEQ ID NO: 26 in which the amino acid at position 132 of SEQ ID NO:
26 is not leucine (L) and the amino acid at position 159 is not
threonine (T), the amino acid at position 132 is cysteine (C), and
the amino acid at position 159 is cysteine (C). In a preferred
embodiment of this isolated polypeptide molecule, the polypeptide
molecule comprises or consists of SEQ ID NO: 16. The invention
provides an isolated nucleic acid molecule that encodes for the
isolated polypeptide comprising or consisting of SEQ ID NO: 16.
Preferably, the isolated nucleic acid molecule that encodes for the
isolated polypeptide comprising or consisting of SEQ ID NO: 16, is
a nucleic acid molecule that comprises or consists of SEQ ID NO:
15.
[0013] In certain embodiments of the isolated polypeptide molecule
comprising or consisting of SEQ ID NO: 26 in which the amino acid
at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino
acid at position 159 is not threonine (T), the amino acid at
position 132 is cysteine (C) and the amino acid at position 159 is
serine(S). The isolated polypeptide molecule comprising or
consisting of SEQ ID NO: 26 in which the amino acid at position 132
of SEQ ID NO: 26 is not leucine (L) and the amino acid at position
159 is not threonine (T), may comprise or consist of SEQ ID NO: 19.
Alternatively, or in addition, the isolated polypeptide molecule
comprising or consisting of SEQ ID NO: 26 in which the amino acid
at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino
acid at position 159 is not threonine (T), wherein the amino acid
at position 132 is cysteine (C) and wherein the amino acid at
position 159 is serine(S) may comprise or consist of SEQ ID NO: 19.
The invention provides an isolated nucleic acid molecule that
encodes for the isolated polypeptide that comprises or consists of
SEQ ID NO: 19. Preferably, the nucleic acid molecule comprises or
consists of SEQ ID NO: 18.
[0014] In certain embodiments of the isolated polypeptide molecule
comprising or consisting of SEQ ID NO: 26 in which the amino acid
at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino
acid at position 159 is not threonine (T), the amino acid at
position 132 is alanine (A) and the amino acid at position 159 is
cysteine (C). The isolated polypeptide molecule comprising or
consisting of SEQ ID NO: 26 in which the amino acid at position 132
of SEQ ID NO: 26 is not leucine (L) and the amino acid at position
159 is not threonine (T) may comprise or consist of SEQ ID NO: 22.
Alternatively, or in addition, the isolated polypeptide molecule
comprising or consisting of SEQ ID NO: 26 in which the amino acid
at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino
acid at position 159 is not threonine (T), wherein the amino acid
at position 132 is alanine (A) and wherein the amino acid at
position 159 is cysteine (C) may comprise or consist of SEQ ID NO:
22. The invention provides an isolated nucleic acid molecule that
encodes for the isolated polypeptide that comprises or consists of
SEQ ID NO: 22. Preferably, this nucleic acid molecule comprises or
consists of SEQ ID NO: 21.
[0015] In certain embodiments of the isolated polypeptide molecule
comprising or consisting of SEQ ID NO: 26 in which the amino acid
at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino
acid at position 159 is not threonine (T), the amino acid at
position 132 is cysteine (C) and the amino acid at position 159 is
alanine (A). The isolated polypeptide molecule comprising or
consisting of SEQ ID NO: 26 in which the amino acid at position 132
of SEQ ID NO: 26 is not leucine (L) and the amino acid at position
159 is not threonine (T) may comprise or consist of SEQ ID NO: 25.
Alternatively, or in addition, the isolated polypeptide molecule
comprising or consisting of SEQ ID NO: 26 in which the amino acid
at position 132 of SEQ ID NO: 26 is not leucine (L) and the amino
acid at position 159 is not threonine (T), wherein the amino acid
at position 132 is cysteine (C) and wherein the amino acid at
position 159 is alanine (A) may comprise or consist of SEQ ID NO:
25. The invention provides an isolated nucleic acid molecule that
encodes for the isolated polypeptide that comprises or consists of
SEQ ID NO: 25. Preferably, this nucleic acid molecule comprises or
consists of SEQ ID NO: 24.
[0016] The invention provides any one of the isolated polypeptide
molecules described herein, wherein the polypeptide molecule
encodes for a mutant Chop2 protein that forms a mutant ChR2, which
elicits a current in response to a threshold intensity of light
that is lower than the threshold of a wild type ChR2 protein.
Moreover, the current conducts cations. Exemplary cations include,
but are not limited to, H.sup.+, Na.sup.+, K.sup.+, and Ca.sup.2+
ions. The ChR2 wild type and mutant proteins described herein
non-specifically conduct cations. Consequently, the current
conducts one or more of the following: H.sup.+, Na.sup.+, K.sup.+,
and Ca.sup.2+ ions.
[0017] The invention provides any one of the isolated polypeptide
molecules described herein further comprising a pharmaceutically
acceptable carrier. The invention also provides a composition
comprising at least one isolated polynucleotide molecule described
herein. The composition may further comprise a
pharmaceutically-acceptable carrier.
[0018] The invention provides an isolated nucleic acid molecule
that encodes for any of the isolated polypeptides described herein.
Moreover, the isolated nucleic acid molecule may further include a
pharmaceutically acceptable carrier. The invention also provides a
composition comprising at least one isolated nucleic acid molecule
described herein. The composition may further comprise a
pharmaceutically-acceptable carrier.
[0019] The invention provides a cell, wherein the cell has been
contacted with or comprises an isolated polypeptide molecule of the
invention. Moreover, the invention provides a cell, wherein the
cell has been contacted with or comprises an isolated nucleic acid
molecule that encodes for an isolated polypeptide molecule of the
invention. The invention provides, a composition comprising,
consisting essentially of, or consisting of a cell that comprises
an isolated polypeptide molecule of the invention or a nucleic acid
molecule that encodes for an isolated polypeptide molecule of the
invention. Cells of the invention may be contacted with the
isolated polypeptide or an isolated nucleic acid encoding the
polypeptide in vitro, ex vivo, in vivo, or in situ. In certain
embodiments of the invention, the cell is a photoreceptor; a
horizontal cell; a bipolar cell; an amacrine cell, and, especially,
an AII amacrine cell; or a retinal ganglion cell, including a
photosensitive retinal ganglion cell. Preferably, the cell is a
retinal ganglion cell, a photosensitive retinal ganglion cell, a
bipolar cell, an ON-type bipolar cell, a rod bipolar cell, or an
AII amacrine cell. In certain aspects of the invention, the cell is
a photoreceptor, a bipolar cell, a rod bipolar cell, an ON-type
cone bipolar cell, a retinal ganglion cell, a photosensitive
retinal ganglion cell, a horizontal cell, an amacrine cell, or an
AII amacrine cell.
[0020] The invention provides a method of improving or restoring
vision, comprising administering to a subject any one of the
compositions described herein. The invention further provides a
prophylactic method of preserving vision, comprising administering
to a subject any one of the compositions described herein.
[0021] The methods described herein may also be applied to those
subjects who are healthy, blind (in part or in total), and/or those
subjects with retinal degeneration (characterized by a loss of rod
and/or cone photoreceptor cells), but may be dependent upon the
activity of photosensitive retinal ganglion cells for a
determination of ambient light levels. For example, the methods
described herein can be used to preserve, improve, or restore the
activity of a photosensitive retinal ganglion cell that mediates
the transduction of light information for synchronizing circadian
rhythms to the 24-hour light/dark cycle, pupillary control and
reflexes, and photic regulation of melatonin release.
[0022] In certain embodiments of the methods of the invention, the
subject may have normal vision or impaired vision. Alternatively,
or in addition, the subject may be at risk for developing an ocular
disease that leads to impairment of vision. For example, the
subject may have a family history of, ocular disease, including,
macular degeneration and retinitis pigmentosa. The subject may be
at risk for incurring an eye injury that causes damage to
photosensitive cells in the retina. The subject may have a genetic
marker or genetic/congenital condition that results in impaired
vision, low vision, legal blindness, partial blindness, or complete
blindness. Subjects may have a refractive defect that results in
myopia (near-sightedness) or hyperopia (far-sightedness).
[0023] Compositions of methods of the invention may be administered
to a subject either systemically or locally. A preferred route of
local administration is intravitreal injection.
[0024] Other features and advantages of the invention will be
apparent from and are encompassed by the following detailed
description and claims.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1A-B show representative recordings of the light-evoked
currents from wild-type (WT) ChR2, L132C, L132C/T159C, and
L132C/159S mutants in HEK cells for comparison of their light
sensitivity (A). The light stimuli (photons/cm.sup.2s at 460 nm)
were generated by a xenon arc lamp and attenuated by neutral
density filters: ND4.0 (2.8.times.10.sup.14), ND3.0
(1.4.times.10.sup.15), ND2.5 (4.8.times.10.sup.15); ND2.0
(1.6.times.10.sup.16), ND1.0 (1.3.times.10.sup.17), ND0
(1.2.times.10.sup.18). The same current traces in FIG. 1A are shown
at a different current scale in FIG. 1B. The traces pointed by
arrows are evoked by the same light intensity (ND2.5).
[0026] FIG. 2 shows representative recordings of the light-evoked
currents from wild-type (WT) ChR2, T159C, L132C, L132C/T159C, and
L132C/T159S mutants to a 10 ms light pulse (1.2.times.10.sup.18
photons/cm.sup.2/s at 460 nm) in HEK cells for comparison of their
deactivation time course (decay time course after light off).
[0027] FIG. 3 shows representative multichannel array recordings of
WT ChR2, L132C, L132C/T159C, and L132C/T159S mediated spiking
activities from retinal ganglion cells in retinal whole-mounts for
comparison of their light sensitivity. Light stimuli
(photons/cm.sup.2/s) was generated by a 473 nm blue laser and
attenuated by neutral density filters: ND0 (6.3.times.10.sup.16),
ND1.0 (7.4.times.10.sup.15), ND1.5 (2.7.times.10.sup.15), ND2.0
(7.3.times.10.sup.14), ND2.5 (3.2.times.10.sup.14), ND3.0
(8.5.times.10.sup.13), ND3.5 (3.8.times.10.sup.13), and ND4.0
(9.5.times.10.sup.12).
[0028] FIG. 4A-B show representative multichannel array recordings
of WT ChR2, L132C, L132C/T159C, and L132C/T159S mediated spiking
activities from retinal ganglion cells in retinal whole-mounts for
comparison of their temporal dynamics. In each panel, the raster
plots of 10 consecutive light-elicited spikes originated from a
single neuron (top) and the averaged spike rate histograms (bottom)
are shown. Light pulses at different frequency was generated by a
473 nm blue laser with intensities about one log unit above the
threshold intensity of each mutant. Recordings of WT ChR2 and L132C
are shown in FIG. 4A, and recordings of L132C/T159C and L132C/T159S
are shown in FIG. 4B.
DETAILED DESCRIPTION
Visual System
[0029] The central nervous system mediates vision (also referred to
herein as sight) through specialized cells and unique methods of
signal transduction present in the visual system. The principle
responsibility of the visual system is to transform light, in the
form of electromagnetic radiation, into a representation or image
of the surrounding world. In addition to the "visual" function of
this system, the visual system also regulates the pupillary light
reflex (PLR), circadian photoentrainment to periodic light/dark
cycles, and release of the hormone melatonin.
[0030] The cells of the retina are the first cells of the visual or
nervous system to encounter light (electromagnetic radiation of
varying wavelengths and intensities). Photons travel through the
cornea, pupil, and lens before reaching the retina. The retina has
a unique structure because the photoreceptor cells that directly
absorb photons are located in the outer layer of the retina.
Photons that traverse the lens first encounter an inner layer of
retinal ganglion cells (a minority of which are photosensitive
through the expression of the opsin, melanopsin) and an
intermediate layer of bipolar cells before reaching the outer layer
of photoreceptor cells (also known as rods and cones). Rod
photoreceptors operate in dim illumination condition (scotopic
vision) while cone photoreceptors operate in bright illumination
conditions (photopic vision) responsible for color vision. Cone
photoreceptors synapse directly onto ON- and OFF-type cone bipolar
cells, which in turn, synapse directly onto ON- and OFF-type
retinal ganglion cells. Rod photoreceptors synapse to rod bipolar
cells (a unique type of bipolar cells, which is ON-type), which
synapse to AII amacrine cells. The AII amacrine cells then relay
the visual signals to ON-type cone bipolar cells through gap
junction and to OFF-type cone bipolar cells as well as OFF ganglion
cells through inhibitory glycinergic synapses. Retinal ganglion
cells are responsible for relating visual information to neurons of
the brain.
Phototransduction
[0031] Within the retina, photoreceptor cells absorb photon
particles and transform the raw data of light frequency and
wavelength into chemical and subsequently electrical signals that
propagate this initial information throughout the visual and
nervous systems. Specifically, an opsin protein located on the
surface of a photoreceptor (rod, cone, and/or photosensitive
retinal ganglion cell) absorbs a photon and initiates an
intracellular signaling cascade, which results in the
hyperpolarization of the photoreceptor. In the dark, the opsin
proteins absorb no photons, the photoreceptors are depolarized. The
visual signals of photoreceptors then relay through bipolar cells,
amacrine cells, and ganglion cells to the high visual centers in
the brain. Specifically, when rod and cone photoreceptors are
depolarized (in the dark), they cause the depolarization of rod
bipolar cells and ON-type cone bipolar cells, but the
hyperpolarization of OFF-type cone bipolar cells, which in turn
cause the depolarization of AII amacrine cells and the increase of
the spiking of ON-type retinal ganglion cells and the decrease of
the spiking of OFF-type retinal ganglion cells. The opposite
happens (to rod, ON- and OFF-bipolar cells, AII amacrine and ON-
and OFF-ganglion cells), when rod and cone photoreceptors are
hyperpolarized (in response to light).
[0032] Light information is processed and refined significantly by
the actions of photoreceptors, bipolar cells, horizontal cells,
amacrine cells, and retinal ganglion cells. To add to the
complexity of this system, photoreceptors are found in three main
varieties, including rods, cones (of which three types respond most
strongly to distinct wavelengths of light), and photosensitive
retinal ganglion cells. Thus, a first layer of information
processing occurs at the level of the photoreceptors which respond
differentially to certain wavelengths and intensities of light.
Bipolar cells of the retina receive information from both
photoreceptor cells and horizontal cells. Horizontal cells of the
retina receive information from multiple photoreceptor cells, and,
therefore, integrate information between cell types and across
distances in the retina. Bipolar cells further integrate
information directly from photoreceptor cells and horizontal cells
by producing mainly graded potentials to retinal ganglion cells,
although some recent studies indicate that some bipolar cells can
generate action potentials. Cone bipolar cells synapse on retinal
ganglion cells and amacrine cells while rod bipolar cells synapse
only to AII amacrine cells. Similar to horizontal cells, most
amacrine cells integrate information laterally within the retina.
Unlike horizontal cells, most amacrine cells are inhibitory
(GABAergic) interneurons. Amacrine cells are also more specialized
than horizontal cells, because each amacrine cell specifically
synapses on a particular type of bipolar cell (one of the ten
varieties of bipolar cell). Particularly, the AII amacrine cell is
a critical relay neuron in the rod pathway (under scotopic vision
when cone photoreceptors do not respond). The AII amacrine cells
receive synaptic inputs from rod bipolar cells and then piggy-back
the signals to cone pathway through ON- and OFF-cone bipolar cells
to ON- and OFF-ganglion cells as described above. Therefore,
expression of Chop2, and the resulting formation of ChR2, in rod
bipolar cells or AII amacrine cells can create both ON and OFF
responses in retinal ganglion cells. Furthermore, retinal ganglion
cells integrate information from bipolar cells and from amacrine
cells. Although retinal ganglion cells vary significantly with
respect to size, connectivity, and responses to visual stimulation
(e.g. visual fields), all retinal ganglion cells extend a long axon
into the brain. Except for a minute portion of the retinal ganglion
cells that transduce non-visual information regarding the pupillary
light reflex and circadian entrainment, the totality of axons
extending from the retinal ganglion cells form the optic nerve,
optic chiasm, and optic tract of the central nervous system.
Consequently, a significant amount of information processing occurs
in the retina itself.
[0033] Photoreceptor cells express endogenous opsin proteins, such
as rhodopsin. The mutant Chop2 proteins of the invention may be
expressed in any cell type, and form functional ChR2 channels.
Preferably, the cell is a retinal cell. Exemplary cells, include,
but are not limited to, photoreceptor cells (e.g., rods, cones, and
photosensitive retinal ganglion cells), horizontal cells, bipolar
cells, amacrine cells, and retinal ganglion cells.
Channelopsin-2 (Chop2)
[0034] Channelopsin-2 (Chop2) was first isolated from the green
algae, Chlamydomonas reinhardtii. Channelopsin-2 is a seven
transmembrane domain protein that becomes photo-switchable (light
sensitive) when bound to the chromophore all-trans-retinal. Chop2,
when linked to a retinal molecule via Schiff base linkage forms a
light-gated, nonspecific, inwardly rectifying, cation channel,
called Channelrhodopsin-2 (Chop2 retinalidene, abbreviated
ChR2).
[0035] As referred to herein, "channelopsin-2" or "Chop2" refers to
the gene that encodes channelopsin-2, which then forms
Channelrhodopsin-2 (ChR2) once bound to retinal. Gene constructs of
the present invention refer primarily to channelopsin-2 (i.e.,
without the retinal), and all Chop2 variants disclosed herein form
functional channelrhodopsin-2 variants. The methods disclosed
herein may include delivering Chop2 to cells without exogenous
retinal. It is understood that upon expression of Chop2 in cells
(i.e., retinal neurons), endogenously available retinal binds to
the wild-type Chop2 or the Chop2 mutants of the present invention
to form functional light-gated channels, WT ChR2 or mutant ChR2. As
such, Chop2 proteins, as referred to herein, can also be synonymous
with ChR2.
[0036] As used herein, "channelrhodopsin-2" or "ChR2" refers to the
retinal-bound functional light-sensitive channel. In one
embodiment, the bound retinal may be provided exogenously. In a
preferred embodiment, the bound retinal is provided from endogenous
levels available in the cell. The present invention also
encompasses the functional channelrhodopsin-2 channels formed by
the polypeptides and polynucleotides encoding the Chop2 mutants
described herein.
[0037] Upon illumination by the preferred dose of light radiation,
ChR2 opens the pore of the channel, through which H.sup.+,
Na.sup.+, K.sup.+, and/or Ca.sup.2+ ions flow into the cell from
the extracellular space. Activation of the ChR2 channel typically
causes a depolarization of the cell expressing the channel.
Depolarized cells produce graded potentials and or action
potentials to carry information from the Chop2/ChR2-expressing cell
to other cells of the retina or brain.
[0038] The wild type form of ChR2 or mutant ChR2s with high
temporal resolution have become a central focus of neuroscience
research. When expressed in a mammalian neuron, ChR2 mediates
light-controlled depolarization of in vitro or ex vivo cultures.
Wild type ChR2s or mutant ChR2s with high temporal resolution (the
latter usually display low light sensitivity) presents several
challenges that must be addressed to enable their use for the
purpose of vision restoration. For the purpose of vision
restoration, the ChR2 with high light sensitivity rather than high
temporal resolution is desired.
[0039] Wild type ChR2 proteins require illumination from high blue
light intensities for full activation (i.e. 10.sup.18-10.sup.19
photons s.sup.-1 cm.sup.-2 at a wavelength of 480 nm). Continuous
illumination of this type can damage cells.
[0040] The kinetics of the wild type ChR2 protein is suboptimal for
maximizing channel efficacy. Efficacy can be increased by modifying
one or more amino acids of the wild type ChR2 protein either to
prolong the open state of the channel or increase the unit
conductance of the channel, or both. The single-channel conductance
of wild-type ChR2 is small. Thus, neuronal activation in vivo would
either require high expression of the wild type channel or very
intense activation with the preferred wavelength of blue-light. A
simpler solution may be found by altering the channel conductance
or to prolong the channel open time. Either one of these mechanisms
and, in particular, the combination of these mechanisms, enable
lower and safer light intensities to be used to achieve the same
level of cellular depolarization.
[0041] For example, mutant ChR2 proteins of the invention achieve
greater light sensitivity through the prolongation of the channel
open state. Consequently, each mutant ChR2 channel conducts a
greater photocurrent than a wild type ChR2 channel when activated
by the same light intensities. Therefore, the mutant channels are
activated by light intensities that are lower than those required
for activation of the wild type ChR2 channels. Quantitatively,
detectable spiking activity of retinal ganglion cells expressing
mutant ChR2 proteins can be elicited by a light intensity that is
1.5-2 log units lower than the light intensity required to elicit
spiking activity from retinal ganglion cells expressing wild type
ChR2. Thus, the light intensities required to activate the mutant
ChR2 proteins are close to or fall within the range of normal
outdoor lighting conditions.
[0042] The following sequences provide non-limiting examples of
wild type and mutant Chop2 proteins, and polynucleotides encoding
said WT and mutant Chop2 proteins of the invention, and forming WT
and mutant ChR2s of the invention.
[0043] A wild type (WT) Chop2 of the invention may be encoded by
the following Chlamydomonas reinhardtii chlamyopsin 4 light-gated
ion channel (COP4) mRNA sequence (GenBank Accession No.
XM_001701673, and SEQ ID NO: 1):
TABLE-US-00001 1 gcagcaccat acttgacatc tgtcgccaag caagcattaa
acatggatta tggaggcgcc 61 ctgagtgccg ttgggcgcga gctgctattt
gtaacgaacc cagtagtcgt caatggctct 121 gtacttgtgc ctgaggacca
gtgttactgc gcgggctgga ttgagtcgcg tggcacaaac 181 ggtgcccaaa
cggcgtcgaa cgtgctgcaa tggcttgctg ctggcttctc catcctactg 241
cttatgtttt acgcctacca aacatggaag tcaacctgcg gctgggagga gatctatgtg
301 tgcgctatcg agatggtcaa ggtgattctc gagttcttct tcgagtttaa
gaacccgtcc 361 atgctgtatc tagccacagg ccaccgcgtc cagtggttgc
gttacgccga gtggcttctc 421 acctgcccgg tcattctcat tcacctgtca
aacctgacgg gcttgtccaa cgactacagc 481 aggcgcacca tgggtctgct
tgtgtctgat attggcacaa ttgtgtgggg cgccacttcc 541 gccatggcca
ccggatacgt caaggtcatc ttcttctgcc tgggtctgtg ttatggtgct 601
aacacgttct ttcacgctgc caaggcctac atcgagggtt accacaccgt gccgaagggc
661 cggtgtcgcc aggtggtgac tggcatggct tggctcttct tcgtatcatg
gggtatgttc 721 cccatcctgt tcatcctcgg ccccgagggc ttcggcgtcc
tgagcgtgta cggctccacc 781 gtcggccaca ccatcattga cctgatgtcg
aagaactgct ggggtctgct cggccactac 841 ctgcgcgtgc tgatccacga
gcatatcctc atccacggcg acattcgcaa gaccaccaaa 901 ttgaacattg
gtggcactga gattgaggtc gagacgctgg tggaggacga ggccgaggct 961
ggcgcggtca acaagggcac cggcaagtac gcctcccgcg agtccttcct ggtcatgcgc
1021 gacaagatga aggagaaggg cattgacgtg cgcgcctctc tggacaacag
caaggaggtg 1081 gagcaggagc aggccgccag ggctgccatg atgatgatga
acggcaatgg catgggtatg 1141 ggaatgggaa tgaacggcat gaacggaatg
ggcggtatga acgggatggc tggcggcgcc 1201 aagcccggcc tggagctcac
tccgcagcta cagcccggcc gcgtcatcct ggcggtgccg 1261 gacatcagca
tggttgactt cttccgcgag cagtttgctc agctatcggt gacgtacgag 1321
ctggtgccgg ccctgggcgc tgacaacaca ctggcgctgg ttacgcaggc gcagaacctg
1381 ggcggcgtgg actttgtgtt gattcacccc gagttcctgc gcgaccgctc
tagcaccagc 1441 atcctgagcc gcctgcgcgg cgcgggccag cgtgtggctg
cgttcggctg ggcgcagctg 1501 gggcccatgc gtgacctgat cgagtccgca
aacctggacg gctggctgga gggcccctcg 1561 ttcggacagg gcatcctgcc
ggcccacatc gttgccctgg tggccaagat gcagcagatg 1621 cgcaagatgc
agcagatgca gcagattggc atgatgaccg gcggcatgaa cggcatgggc 1681
ggcggtatgg gcggcggcat gaacggcatg ggcggcggca acggcatgaa caacatgggc
1741 aacggcatgg gcggcggcat gggcaacggc atgggcggca atggcatgaa
cggaatgggt 1801 ggcggcaacg gcatgaacaa catgggcggc aacggaatgg
ccggcaacgg aatgggcggc 1861 ggcatgggcg gcaacggtat gggtggctcc
atgaacggca tgagctccgg cgtggtggcc 1921 aacgtgacgc cctccgccgc
cggcggcatg ggcggcatga tgaacggcgg catggctgcg 1981 ccccagtcgc
ccggcatgaa cggcggccgc ctgggtacca acccgctctt caacgccgcg 2041
ccctcaccgc tcagctcgca gctcggtgcc gaggcaggca tgggcagcat gggaggcatg
2101 ggcggaatga gcggaatggg aggcatgggt ggaatggggg gcatgggcgg
cgccggcgcc 2161 gccacgacgc aggctgcggg cggcaacgcg gaggcggaga
tgctgcagaa tctcatgaac 2221 gagatcaatc gcctgaagcg cgagcttggc
gagtaaaagg ctggaggccg gtactgcgat 2281 acctgcgagc tcgcgcgcct
gactcgtcgt acacacggct caggagcacg cgcgcgtgga 2341 cttctcaacc
tgtgtgcaac gtatctagag cggcctgtgc gcgaccgtcc gtgagcattc 2401
cggtgcgatc ttcccgcctt cgcaccgcaa gttcccttcc tggccctgct gcgcctgacg
2461 catcgtccga acggaagggc ggcttgatca gtaaagcatt gaagactgaa
gtcgtgcgac 2521 cgtagtgcta tggctctgca cgtaagtggg cgctgccctg
cttactacgc attgcccaag 2581 actgcttcct tttggtggcc gaggccctgg
tcccacatca ttcatttgca taacgtactg 2641 tttagttaca tacgctttgc
ttaacctcga caattgcaac atgggctgag agtccgtacg 2701 gcggctatgg
acgaaggtgt tatcggatgt gattaggaat ctcggttgaa aggcttcgag 2761
aaagtgagct tcatctgtgg cttctgttgg ggtcatcaag aagaacgacg gtaaggcaaa
2821 cgaggtaaaa gtggcacgtc tttgtgcaca acgggcccgt ggagagtggg
ggagtgcatg 2881 tgtgcggtcc taacacgcga gtgcaaagcg ggcttttctg
gagctgggtt acggtctggc 2941 tcggcaactg ctctgtgttt taaccacagc
ttcggaagtc tgggtatgtt ttgttggcag 3001 aaacatttgg gtaacttgag
ggtgattcgt ctggagtcgg acaacatggc tgccgtccgt 3061 gtgcagggac
ggtaatcaat gagctggagc tgtgatgctc accacacgtt gcatacccct 3121
gcttacaaaa acactttgat gtcgtggcca aactatgcgt gagcaaagag ttaaagaggc
3181 atgagtgcat ggttgcggac gtgcgcaaca attgcatcaa gtatttgacg
ccttcaagcc 3241 aacaagtgcg cgcgcggcaa cttgattaac acgccggacg
cagtggtggg ggcgtgtaca 3301 gtgtttatga gctgccattc tgcgatccgt
agtgttaggt tgcgtgtgac gccgcgcggc 3361 tgtgggccct tacatggaga
gttgggtgct tcaccacacg gttggcgccg ctgaagggtg 3421 tgctatgttt
tggtaaagcc ggggccctga agaccgcaac cgtagaaccg tactgaaagg 3481
gtgtcagccc ggggtaactg gatgccctgg gacatagcta ttaatgttga agtgaagccg
3541 agccgag tgccgtgcgc cgctgtatca ccaaggcccg tccta
[0044] A wild type (WT) ChR2 of the invention may be encoded by the
following Chlamydomonas reinhardtii chlamyopsin 4 light-gated ion
channel (COP4) amino acid sequence (GenBank Accession No.
XP_001701725, and SEQ ID NO: 2):
TABLE-US-00002 1 mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi
esrgtngaqt asnvlqwlaa 61 gfsilllmfy ayqtwkstcg weeiyvcaie
mvkvilefff efknpsmlyl atghrvqwlr 121 yaewlltcpv ilihlsnltg
lsndysrrtm gllvsdigti vwgatsamat gyvkviffcl 181 glcygantff
haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl 241
svygstvght iidlmskncw gllghylrvl ihehilihgd irkttklnig gteievetlv
301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq
aaraammmmn 361 gngmgmgmgm ngmngmggmn gmaggakpgl eltpqlqpgr
vilavpdism vdffreqfaq 421 lsvtyelvpa lgadntlalv tqaqnlggvd
fvlihpeflr drsstsilsr lrgagqrvaa 481 fgwaqlgpmr dliesanldg
wlegpsfgqg ilpahivalv akmqqmrkmq qmqqigmmtg 541 gmngmgggmg
ggmngmgggn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma 601
gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggmm nggmaapqsp gmnggrlgtn
661 plfnaapspl ssqlgaeagm gsmggmggms gmggmggmgg mggagaattq
aaggnaeaem 721 nlmneinr lkrelge
[0045] A wild type (WT) Chop2 of the invention may be encoded by
the following Chlamydomonas reinhardtii retinal binding protein
(cop4) gene sequence (GenBank Accession No. AF461397, and SEQ ID
NO: 3):
TABLE-US-00003 1 gcatctgtcg ccaagcaagc attaaacatg gattatggag
gcgccctgag tgccgttggg 61 cgcgagctgc tatttgtaac gaacccagta
gtcgtcaatg gctctgtact tgtgcctgag 121 gaccagtgtt actgcgcggg
ctggattgag tcgcgtggca caaacggtgc ccaaacggcg 181 tcgaacgtgc
tgcaatggct tgctgctggc ttctccatcc tactgcttat gttttacgcc 241
taccaaacat ggaagtcaac ctgcggctgg gaggagatct atgtgtgcgc tatcgagatg
301 gtcaaggtga ttctcgagtt cttcttcgag tttaagaacc cgtccatgct
gtatctagcc 361 acaggccacc gcgtccagtg gttgcgttac gccgagtggc
ttctcacctg cccggtcatt 421 ctcattcacc tgtcaaacct gacgggcttg
tccaacgact acagcaggcg caccatgggt 481 ctgcttgtgt ctgatattgg
cacaattgtg tggggcgcca cttccgccat ggccaccgga 541 tacgtcaagg
tcatcttctt ctgcctgggt ctgtgttatg gtgctaacac gttctttcac 601
gctgccaagg cctacatcga gggttaccac accgtgccga agggccggtg tcgccaggtg
661 gtgactggca tggcttggct cttcttcgta tcatggggta tgttccccat
cctgttcatc 721 ctcggccccg agggcttcgg cgtcctgagc gtgtacggct
ccaccgtcgg ccacaccatc 781 attgacctga tgtcgaagaa ctgctggggt
ctgctcggcc actacctgcg cgtgctgatc 841 cacgagcata tcctcatcca
cggcgacatt cgcaagacca ccaaattgaa cattggtggc 901 actgagattg
aggtcgagac gctggtggag gacgaggccg aggctggcgc ggtcaacaag 961
ggcaccggca agtacgcctc ccgcgagtcc ttcctggtca tgcgcgacaa gatgaaggag
1021 aagggcattg acgtgcgcgc ctctctggac aacagcaagg aggtggagca
ggagcaggcc 1081 gccagggctg ccatgatgat gatgaacggc aatggcatgg
gtatgggaat gggaatgaac 1141 ggcatgaacg gaatgggcgg tatgaacggg
atggctggcg gcgccaagcc cggcctggag 1201 ctcactccgc agctacagcc
cggccgcgtc atcctggcgg tgccggacat cagcatggtt 1261 gacttcttcc
gcgagcagtt tgctcagcta tcggtgacgt acgagctggt gccggccctg 1321
ggcgctgaca acacactggc gctggttacg caggcgcaga acctgggcgg cgtggacttt
1381 gtgttgattc accccgagtt cctgcgcgac cgctctagca ccagcatcct
gagccgcctg 1441 cgcggcgcgg gccagcgtgt ggctgcgttc ggctgggcgc
agctggggcc catgcgtgac 1501 ctgatcgagt ccgcaaacct ggacggctgg
ctggagggcc cctcgttcgg acagggcatc 1561 ctgccggccc acatcgttgc
cctggtggcc aagatgcagc agatgcgcaa gatgcagcag 1621 atgcagcaga
ttggcatgat gaccggcggc atgaacggca tgggcggcgg tatgggcggc 1681
ggcatgaacg gcatgggcgg cggcaacggc atgaacaaca tgggcaacgg catgggcggc
1741 ggcatgggca acggcatggg cggcaatggc atgaacggaa tgggtggcgg
caacggcatg 1801 aacaacatgg gcggcaacgg aatggccggc aacggaatgg
gcggcggcat gggcggcaac 1861 ggtatgggtg gctccatgaa cggcatgagc
tccggcgtgg tggccaacgt gacgccctcc 1921 gccgccggcg gcatgggcgg
catgatgaac ggcggcatgg ctgcgcccca gtcgcccggc 1981 atgaacggcg
gccgcctggg taccaacccg ctcttcaacg ccgcgccctc accgctcagc 2041
tcgcagctcg gtgccgaggc aggcatgggc agcatgggag gcatgggcgg aatgagcgga
2101 atgggaggca tgggtggaat ggggggcatg ggcggcgccg gcgccgccac
gacgcaggct 2161 gcgggcggca acgcggaggc ggagatgctg cagaatctca
tgaacgagat caatcgcctg 2221 cgcgagc ttggcgagta a
[0046] A wild type (WT) Chop2 of the invention may be encoded by
the following Chlamydomonas reinhardtii retinal binding protein
(cop4) amino acid sequence (GenBank Accession No. AAM15777, and SEQ
ID NO: 4):
TABLE-US-00004 1 mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi
esrgtngaqt asnvlqwlaa 61 gfsilllmfy ayqtwkstcg weeiyvcaie
mvkvilefff efknpsmlyl atghrvqwlr 121 yaewlltcpv ilihlsnltg
lsndysrrtm gllvsdigti vwgatsamat gyvkviffcl 181 glcygantff
haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl 241
svygstvght iidlmskncw gllghylrvl ihehilihgd irkttklnig gteievetlv
301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq
aaraammmmn 361 gngmgmgmgm ngmngmggmn gmaggakpgl eltpqlqpgr
vilavpdism vdffreqfaq 421 lsvtyelvpa lgadntlalv tqaqnlggvd
fvlihpeflr drsstsilsr lrgagqrvaa 481 fgwaqlgpmr dliesanldg
wlegpsfgqg ilpahivalv akmqqmrkmq qmqqigmmtg 541 gmngmgggmg
ggmngmgggn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma 601
gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggmm nggmaapqsp gmnggrlgtn
661 plfnaapspl ssqlgaeagm gsmggmggms gmggmggmgg mggagaattq
aaggnaeaem 721 nlmneinr lkrelge
[0047] A wild type (WT) Chop2 of the invention may be encoded by
the following Chlamydomonas reinhardtii sensory opsin B (CSOB) mRNA
sequence (GenBank Accession No. AF508966, and SEQ ID NO: 5):
TABLE-US-00005 1 ttgacatctg tcgccaagca agcattaaac atggattatg
gaggcgccct gagtgccgtt 61 gggcgcgagc tgctatttgt aacgaaccca
gtagtcgtca atggctctgt acttgtgcct 121 gaggaccagt gttactgcgc
gggctggatt gagtcgcgtg gcacaaacgg tgcccaaacg 181 gcgtcgaacg
tgctgcaatg gcttgctgct ggcttctcca tcctactgct tatgttttac 241
gcctaccaaa catggaagtc aacctgcggc tgggaggaga tctatgtgtg cgctatcgag
301 atggtcaagg tgattctcga gttcttcttc gagtttaaga acccgtccat
gctgtatcta 361 gccacaggcc accgcgtcca gtggttgcgt tacgccgagt
ggcttctcac ctgcccggtc 421 attctcattc acctgtcaaa cctgacgggc
ttgtccaacg actacagcag gcgcaccatg 481 ggtctgcttg tgtctgatat
tggcacaatt gtgtggggcg ccacttccgc catggccacc 541 ggatacgtca
aggtcatctt cttctgcctg ggtctgtgtt atggtgctaa cacgttcttt 601
cacgctgcca aggcctacat cgagggttac cacaccgtgc cgaagggccg gtgtcgccag
661 gtggtgactg gcatggcttg gctcttcttc gtatcatggg gtatgttccc
catcctgttc 721 atcctcggcc ccgagggctt cggcgtcctg agcgtgtacg
gctccaccgt cggccacacc 781 atcattgacc tgatgtcgaa gaactgctgg
ggtctgctcg gccactacct gcgcgtgctg 841 atccacgagc atatcctcat
ccacggcgac attcgcaaga ccaccaaatt gaacattggt 901 ggcactgaga
ttgaggtcga gacgctggtg gaggacgagg ccgaggctgg cgcggtcaac 961
aagggcaccg gcaagtacgc ctcccgcgag tccttcctgg tcatgcgcga caagatgaag
1021 gagaagggca ttgacgtgcg cgcctctctg gacaacagca aggaggtgga
gcaggagcag 1081 gccgccaggg ctgccatgat gatgatgaac ggcaatggca
tgggtatggg aatgggaatg 1141 aacggcatga acggaatggg cggtatgaac
gggatggctg gcggcgccaa gcccggcctg 1201 gagctcactc cgcagctaca
gcccggccgc gtcatcctgg cggtgccgga catcagcatg 1261 gttgacttct
tccgcgagca gtttgctcag ctatcggtga cgtacgagct ggtgccggcc 1321
ctgggcgctg acaacacact ggcgctggtt acgcaggcgc agaacctggg cggcgtggac
1381 tttgtgttga ttcaccccga gttcctgcgc gaccgctcta gcaccagcat
cctgagccgc 1441 ctgcgcggcg cgggccagcg tgtggctgcg ttcggctggg
cgcagctggg gcccatgcgt 1501 gacctgatcg agtccgcaaa cctggacggc
tggctggagg gcccctcgtt cggacagggc 1561 atcctgccgg cccacatcgt
tgccctggtg gccaagatgc agcagatgcg caagatgcag 1621 cagatgcagc
agattggcat gatgaccggc ggcatgaacg gcatgggcgg cggtatgggc 1681
ggcggcatga acggcatggg cggcggcaac ggcatgaaca acatgggcaa cggcatgggc
1741 ggcggcatgg gcaacggcat gggcggcaat ggcatgaacg gaatgggtgg
cggcaacggc 1801 atgaacaaca tgggcggcaa cggaatggcc ggcaacggaa
tgggcggcgg catgggcggc 1861 aacggtatgg gtggctccat gaacggcatg
agctccggcg tggtggccaa cgtgacgccc 1921 tccgccgccg gcggcatggg
cggcatgatg aacggcggca tggctgcgcc ccagtcgccc 1981 ggcatgaacg
gcggccgcct gggtaccaac ccgctcttca acgccgcgcc ctcaccgctc 2041
agctcgcagc tcggtgccga ggcaggcatg ggcagcatgg gaggcatggg cggaatgagc
2101 ggaatgggag gcatgggtgg aatggggggc atgggcggcg ccggcgccgc
cacgacgcag 2161 gctgcgggcg gcaacgcgga ggcggagatg ctgcagaatc
tcatgaacga gatcaatcgc 2221 ctgaagcgcg agcttggcga gtaaaaggct
ggaggccggt actgcgatac ctgcgagctc 2281 gcgcgcctga ctcgtcgtac
acacggctca ggagcacgcg cgcgtggact tctcaacctg 2341 tgtgcaacgt
atctagagcg gcctgtgcgc gaccgtccgt gagcattccg gtgcgatctt 2401
cccgccttcg caccgcaagt tcccttcctg gccctgctgc gcctgacgca tcgtccgaac
2461 ggaagggcgg cttgatcagt aaagcattga agactgaagt cgtgcgaccg
tagtgctatg 2521 gctctgcacg taagtgggcg ctgccctgct tactacgcat
tgcccaagac tgcttccttt 2581 tggtggccga ggccctggtc ccacatcatt
catttgcata acgtactgtt tagttacata 2641 cgctttgctt aacctcgaca
attgcaacat gggctgagag tccgtacggc ggctatggac 2701 gaaggtgtta
tcggatgtga ttaggaatct cggttgaaag gcttcgagaa agtgagcttc 2761
ttctgtggct tctgttgggg tcatcaagaa gaacgacggt aaggcaaacg aggtaaaagt
2821 ggcacgtctt tgtgcacaac gggcccgtgg agagtggggg agtgcatgtg
tgcggtccta 2881 acacgcgagt gcaaagcggg cttttctgga gctgggttac
ggtctggctc ggcaactgct 2941 ctgtgtttta accacagctt cggaagtctg
ggtatgtttt gttggcagaa acatttgggt 3001 aacttgaggg tgattcgtct
ggagtcggac aacatggctg ccgtccgtgt gcagggacgg 3061 taatcaatga
agctgaagct gtgatgctca ccacacgttg catacccctg cttacaaaaa 3121
cactttgatg tcgtggccaa actatgcgtg agcaaagagt taaagaggca tgagtgcatg
3181 gttgcggacg tgcgcaacaa ttgcatcaag tatttgacgc cttcaagcca
acaagtgcgc 3241 gcgcggcaac ttgattaaca cgccggacgc agtggtgggg
gcgtgtacag tgtttatgag 3301 ctgccattct gcgatccgta gtgttaggtt
gcgtgtgacg ccgcgcggct gtgggccctt 3361 acatggagag ttgggtgctt
caccacacgg ttggcgccgc tgaagggtgt gctatgtttt 3421 ggtaaagccg
gggccctgaa gaccgcaacc gtagaaccgt actgaaaggg tgtcagcccg 3481
gggtaactgg atgccctggg acatagctat taatgttgaa gtgaagccgt caagccgagt
3541 gccgtgcgcc gctgtatcac caaggcccgt ccaaaaaaaa aaaaaaaaaa
aaaaaaaaa
[0048] A wild type (WT) Chop2 of the invention may be encoded by
the following Chlamydomonas reinhardtii sensory opsin B (CSOB)
amino acid sequence (GenBank Accession No. AAM44040, and SEQ ID NO:
6):
TABLE-US-00006 1 mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi
esrgtngaqt asnvlqwlaa 61 gfsilllmfy ayqtwkstcg weeiyvcaie
mvkvilefff efknpsmlyl atghrvqwlr 121 yaewlltcpv ilihlsnitg
lsndysrrtm gllvsdigti vwgatsamat gyvkviffcl 181 glcygantff
haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl 241
svygstvght iidlmskncw gllghylrvl ihehilihgd irkttklnig gteievetlv
301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq
aaraammmmn 361 gngmgmgmgm ngmngmggmn gmaggakpgl eltpqlqpgr
vilavpdism vdffreqfaq 421 lsvtyelvpa lgadntlalv tqaqnlggvd
fvlihpeflr drsstsilsr lrgagqrvaa 481 fgwaqlgpmr dliesanldg
wlegpsfgqg ilpahivalv akmqqmrkmq qmqqigmmtg 541 gmngmgggmg
ggmngmgggn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma 601
gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggmm nggmaapqsp gmnggrlgtn
661 plfnaapspl ssqlgaeagm gsmggmggms gmggmggmgg mggagaattq
aaggnaeaem 721 lqnlmneinr lkrelge
[0049] A wild type (WT) Chop2 of the invention may be encoded by
the following Chlamydomonas reinhardtii acop2 mRNA for
archaeal-type opsin 2 nucleic acid sequence (GenBank Accession No.
AB058891, and SEQ ID NO: 7):
TABLE-US-00007 1 catctgtcgc caagcaagca ttaaacatgg attatggagg
cgccctgagt gccgttgggc 61 gcgagctgct atttgtaacg aacccagtag
tcgtcaatgg ctctgtactt gtgcctgagg 121 accagtgtta ctgcgcgggc
tggattgagt cgcgtggcac aaacggtgcc caaacggcgt 181 cgaacgtgct
gcaatggctt gctgctggct tctccatcct actgcttatg ttttacgcct 241
accaaacatg gaagtcaacc tgcggctggg aggagatcta tgtgtgcgct atcgagatgg
301 tcaaggtgat tctcgagttc ttcttcgagt ttaagaaccc gtccatgctg
tatctagcca 361 caggccaccg cgtccagtgg ttgcgttacg ccgagtggct
tctcacctgc ccggtcattc 421 tcattcacct gtcaaacctg acgggcttgt
ccaacgacta cagcaggcgc accatgggtc 481 tgcttgtgtc tgatattggc
acaattgtgt ggggcgccac ttccgccatg gccaccggat 541 acgtcaaggt
catcttcttc tgcctgggtc tgtgttatgg tgctaacacg ttctttcacg 601
ctgccaaggc ctacatcgag ggttaccaca ccgtgccgaa gggccggtgt cgccaggtgg
661 tgactggcat ggcttggctc ttcttcgtat catggggtat gttccccatc
ctgttcatcc 721 tcggccccga gggcttcggc gtcctgagcg tgtacggctc
caccgtcggc cacaccatca 781 ttgacctgat gtcgaagaac tgctggggtc
tgctcggcca ctacctgcgc gtgctgatcc 841 acgagcatat cctcatccac
ggcgacattc gcaagaccac caaattgaac attggtggca 901 ctgagattga
ggtcgagacg ctggtggagg acgaggccga ggctggcgcg gtcaacaagg 961
gcaccggcaa gtacgcctcc cgcgagtcct tcctggtcat gcgcgacaag atgaaggaga
1021 agggcattga cgtgcgcgcc tctctggaca acagcaagga ggtggagcag
gagcaggccg 1081 ccagggctgc catgatgatg atgaacggca atggcatggg
tatgggaatg ggaatgaacg 1141 gcatgaacgg aatgggcggt atgaacggga
tggctggcgg cgccaagccc ggcctggagc 1201 tcactccgca gctacagccc
ggccgcgtca tcctggcggt gccggacatc agcatggttg 1261 acttcttccg
cgagcagttt gctcagctat cggtgacgta cgagctggtg ccggccctgg 1321
gcgctgacaa cacactggcg ctggttacgc aggcgcagaa cctgggcggc gtggactttg
1381 tgttgattca ccccgagttc ctgcgcgacc gctctagcac cagcatcctg
agccgcctgc 1441 gcggcgcggg ccagcgtgtg gctgcgttcg gctgggcgca
gctggggccc atgcgtgacc 1501 tgatcgagtc cgcaaacctg gacggctggc
tggagggccc ctcgttcgga cagggcatcc 1561 tgccggccca catcgttgcc
ctggtggcca agatgcagca gatgcgcaag atgcagcaga 1621 tgcagcagat
tggcatgatg accggcggca tgaacggcat gggcggcggt atgggcggcg 1681
gcatgaacgg catgggcggc ggcaacggca tgaacaacat gggcaacggc atgggcggcg
1741 gcatgggcaa cggcatgggc ggcaatggca tgaacggaat gggtggcggc
aacggcatga 1801 acaacatggg cggcaacgga atggccggca acggaatggg
cggcggcatg ggcggcaacg 1861 gtatgggtgg ctccatgaac ggcatgagct
ccggcgtggt ggccaacgtg acgccctccg 1921 ccgccggcgg catgggcggc
atgatgaacg gcggcatggc tgcgccccag tcgcccggca 1981 tgaacggcgg
ccgcctgggt accaacccgc tcttcaacgc cgcgccctca ccgctcagct 2041
cgcagctcgg tgccgaggca ggcatgggca gcatgggagg catgggcgga atgagcggaa
2101 tgggaggcat gggtggaatg gggggcatgg gcggcgccgg cgccgccacg
acgcaggctg 2161 cgggcggcaa cgcggaggcg gagatgctgc agaatctcat
gaacgagatc aatcgcctga 2221 agcgcgagct tggcgagtaa aaggctggag
gccggtactg cgatacctgc gagctcgcgc 2281 gcctgactcg tcgtacacac
ggctcaggag cacgcgcgcg tggacttctc aacctgtgtg 2341 caacgtatct
agagcggcct gtgcgcgacc gtccgtgagc attccggtgc gatcttcccg 2401
ccttcgcacc gcaagttccc ttcctggccc tgctgcgcct gacgcatc
[0050] A wild type (WT) Chop2 of the invention may be encoded by
the following Chlamydomonas reinhardtii acop2 mRNA for
archaeal-type opsin 2 amino acid sequence (GenBank Accession No.
BAB68567, and SEQ ID NO: 8):
TABLE-US-00008 1 mdyggalsav grellfvtnp vvvngsvlvp edqcycagwi
esrgtngaqt asnvlqwlaa 61 gfsilllmfy ayqtwkstcg weeiyvcaie
mvkvilefff efknpsmlyl atghrvqwlr 121 yaewlltcpv ilihlsnitg
lsndysrrtm gllvsdigti vwgatsamat gyvkviffcl 181 glcygantff
haakayiegy htvpkgrcrq vvtgmawlff vswgmfpilf ilgpegfgvl 241
svygstvght iidlmskncw gllghylrvl ihehilihgd irkttklnig gteievetlv
301 edeaeagavn kgtgkyasre sflvmrdkmk ekgidvrasl dnskeveqeq
aaraammmmn 361 gngmgmgmgm ngmngmggmn gmaggakpgl eltpqlqpgr
vilavpdism vdffreqfaq 421 lsvtyelvpa lgadntlalv tqaqnlggvd
fvlihpeflr drsstsilsr lrgagqrvaa 481 fgwaqlgpmr dliesanldg
wlegpsfgqg ilpahivalv akmqqmrkmq qmqqigmmtg 541 gmngmgggmg
ggmngmgggn gmnnmgngmg ggmgngmggn gmngmgggng mnnmggngma 601
gngmgggmgg ngmggsmngm ssgvvanvtp saaggmggmm nggmaapqsp gmnggrlgtn
661 plfnaapspl ssqlgaeagm gsmggmggms gmggmggmgg mggagaattq
aaggnaeaem 721 lqnlmneinr lkrelge
ChR2 Mutants
[0051] The present invention provides Chop2 mutants wherein one or
more amino acids are mutated. In some embodiments, the Chop2 is the
full-length polypeptide, such as SEQ ID NOs: 2, 4, 6, and 8, with
at least one amino acid mutation. In some embodiments, the mutation
is at amino acid 132 and/or amino acid 159. In some preferred
embodiments, the amino acid at position 132 is mutated from a
leucine to a cysteine or an alanine. In some preferred embodiments,
the amino acid at position 159 is mutated from a threonine to an
alanine, a cysteine, or a serine. In all embodiments, the Chop2
mutants form a functional ChR2 channel.
[0052] The present invention also encompasses Chop2 proteins and
nucleic acids that encode a biologically active fragment or a
conservative amino acid substitution or other mutation variant of
Chop2. Non-limiting examples of useful fragments include
polypeptides encoding amino acids 1-315 of the wild-type Chop2,
i.e., SEQ ID NO: 26, wherein at least one amino acid is mutated or
conservatively substituted, for example at amino acid positions 132
and/or 159. Smaller fragments of wild-type Chop2, wherein at least
one amino acid is mutated or conservatively substituted (i.e., at
amino acid positions 132 and/or 159) may also be useful in the
present invention. Accordingly, Chop2 polypeptides and nucleic
acids of the present invention further include, but are not limited
to, biologically active fragments encoding amino acids 1-315,
1-310, 1-300, 1-275, 1-250, 1-225, 1-200, 1-175, or 1-160 of the
wild-type Chop2, wherein at least one amino acid is mutated or
conservatively substituted, for example at amino acid positions 132
and/or 159. In other embodiments, the Chop2 polypeptides and
nucleic acids of the present invention can be up to, or about, 315
amino acids long, 310 amino acids long, 300 amino acids long, 275
amino acids long, 250 amino acids long, 225 amino acids long, 200
amino acids long, 175 amino acids long, or 160 amino acids
long.
[0053] A single mutant Chop2 of the invention may be encoded by the
following Synthetic construct hVChR1-mKate-betahChR2(L132C) gene
sequence (GenBank Accession No. JN836746, and SEQ ID NO: 9) with
the following annotations, GFP sequence is in bold, L132C Chop2
sequence is underlined:
TABLE-US-00009 1 atggattacc ctgtggcccg gtccctgatt gtaagatacc
ccaccgatct gggcaatgga 61 accgtgtgca tgcccagagg acaatgctac
tgcgaggggt ggctgaggag ccggggcact 121 agtatcgaaa aaaccatcgc
tatcaccctc cagtgggtag tgttcgctct gtccgtagcc 181 tgtctcggct
ggtatgcata ccaagcctgg agggctacct gtgggtggga ggaagtatac 241
gtggccctga tcgagatgat gaagtccatc atcgaggctt tccatgagtt cgactcccca
301 gccacactct ggctcagcag tgggaatggc gtagtgtgga tgagatatgg
agagtggctg 361 ctgacctgtc ccgtcctgct cattcatctg tccaatctga
ccgggctgaa agatgactac 421 tccaagagaa caatgggact gctggtgagt
gacgtggggt gtattgtgtg gggagccacc 481 tccgccatgt gcactggatg
gaccaagatc ctctttttcc tgatttccct ctcctatggg 541 atgtatacat
acttccacgc cgctaaggtg tatattgagg ccttccacac tgtacctaaa 601
ggcatctgta gggagctcgt gcgggtgatg gcatggacct tctttgtggc ctgggggatg
661 ttccccgtgc tgttcctcct cggcactgag ggatttggcc acattagtcc
ttacgggtcc 721 gcaattggac actccatcct ggatctgatt gccaagaata
tgtggggggt gctgggaaat 781 tatctgcggg taaagatcca cgagcatatc
ctgctgtatg gcgatatcag aaagaagcag 841 aaaatcacca ttgctggaca
ggaaatggag gtggagacac tggtagcaga ggaggaggac 901 gggaccgcgg
tcgccaccat ggtgtctaag ggcgaagagc tgattaagga gaacatgcac 961
atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc
1021 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
gggcggccct 1081 ctccccttcg ccttcgacat cctggctacc agcttcatgt
acggcagcaa aaccttcatc 1141 aaccacaccc agggcatccc cgacttcttt
aagcagtcct tccctgaggg cttcacatgg 1201 gagagagtca ccacatacga
agacgggggc gtgctgaccg ctacccagga caccagcctc 1261 caggacggct
gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc 1321
cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagatgct gtaccccgct
1381 gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg
gggccacctg 1441 atctgcaact tgaagaccac atacagatcc aagaaacccg
ctaagaacct caagatgccc 1501 ggcgtctact atgtggacag aagactggaa
agaatcaagg aggccgacaa agagacctac 1561 gtcgagcagc acgaggtggc
tgtggccaga tactgcgacc tccctagcaa actggggcac 1621 aaacttaatt
gcctgcagga gaagaagtca tgcagccagc gcatggccga attccggcaa 1681
tactgttgga acccggacac tgggcagatg ctgggccgca ccccagcccg gtgggtgtgg
1741 atcagcctgt actatgcagc tttctacgtg gtcatgactg ggctctttgc
cttgtgcatc 1801 tatgtgctga tgcagaccat tgatccctac acccccgact
accaggacca gttaaagtca 1861 ccgggggtaa ccttgagacc ggatgtgtat
ggggaaagag ggctgcagat ttcctacaac 1921 atctctgaaa acagctctag
acaggcccag atcaccggac gtccggagac tgagacattg 1981 ccaccggtgg
actacggggg ggccctgagc gctgtgggca gagaactcct gttcgtgaca 2041
aatccagtcg tggtgaacgg ctccgtactc gtacccgagg atcagtgcta ttgcgcagga
2101 tggatcgaga gcagaggcac aaacggcgca cagactgcat ccaacgtgct
ccagtggttg 2161 gccgcaggct tttccattct cctgctcatg ttttacgcct
accagacttg gaagtccaca 2221 tgtggctggg aggaaatcta cgtgtgtgca
atcgaaatgg tgaaggtgat cctggagttt 2281 ttcttcgaat ttaaaaaccc
aagcatgctg tacctggcta ctggccacag agtgcagtgg 2341 ctgcggtatg
ccgaatggct gctgacttgc ccagtgattt gcatccacct gtccaacctg 2401
actgggctgt ctaacgatta cagtaggaga acaatgggac tgctcgtatc cgacatcggc
2461 actatcgtat ggggcgcaac tagtgccatg gccactggat acgtgaaagt
gatcttcttc 2521 tgcctgggac tctgctacgg agcaaacaca ttttttcatg
ccgcaaaagc atatatcgag 2581 gggtatcata ccgtcccaaa gggccggtgt
agacaagtgg tgactggcat ggcttggctg 2641 ttcttcgtgt cctgggggat
gtttcccatc ctctttatcc tgggcccaga aggcttcggg 2701 gtgctgagtg
tgtatggcag taccgtagga cacactatca ttgacctgat gagcaaaaac 2761
tgctgggggc tgctcggcca ctacctgaga gtactcatcc acgagcatat cctgattcat
2821 ggcgatatcc ggaaaactac caagctcaat atcgggggca ccgagattga
agtggagaca 2881 ctcgtggagg acgaggccga ggccggagca gtgaacaaag
gcactggcaa gtatgcctcc 2941 agagaatcct ttctggtgat gcgggacaaa
atgaaggaga aaggcattga tgtacggtgc 3001 agtaatgcca aagccgtcga
gactgatgtg tag
[0054] A single mutant ChR2 of the invention may be encoded by the
following Synthetic construct hVChR1-mKate-betahChR2(L132C) amino
acid sequence (GenBank Accession No. AER29839, and SEQ ID NO: 10)
with the following annotations, GFP sequence is in bold, L132C
Chop2 sequence is underlined:
TABLE-US-00010 1 mdypvarsli vryptdlgng tvcmprgqcy cegw1rsrgt
siektiaitl qwvvfalsva 61 clgwyayqaw ratcgweevy valiemmksi
ieafhefdsp atlwlssgng vvwmrygewl 121 ltcpvllihl snitglkddy
skrtmgllvs dvgcivwgat samctgwtki lfflislsyg 181 mytyfhaakv
yieafhtvpk gicrelvrvm awtffvawgm fpvlfllgte gfghispygs 241
aighsildli aknmwgvlgn ylrvkihehi llygdirkkq kitiagqeme vetivaeeed
301 gtavatmvsk geelikenmh mklymegtvn nhhfkctseg egkpyegtqt
mrikvveggp 361 lpfafdilat sfmygsktfi nhtqgipdff kqsfpegftw
ervttyedgg vltatqdtsl 421 qdgcllynvk irgvnfpsng pvmqkktlgw
eastemlypa dgglegradm alklvggghl 481 icnlkttyrs kkpaknlkmp
gvyyvdrrle rikeadkety veqhevavar ycdlpsklgh 541 klnclqekks
csqrmaefrq ycwnpdtgqm lgrtparwvw islyyaafyv vmtglfalci 601
yvlmqtidpy tpdyqdqlks pgvtlrpdvy gerglqisyn isenssrqaq itgrpetetl
661 ppvdyggals avgrellfvt npvvvngsvl vpedqcycag wiesrgtnga
qtasnvlqwl 721 aagfsilllm fyayqtwkst cgweeiyvca iemvkvilef
ffefknpsml ylatghrvqw 781 lryaewlltc pvicihlsnl tglsndysrr
tmgllvsdig tivwgatsam atgyvkviff 841 clglcygant ffhaakayie
gyhtvpkgrc rqvvtgmawl ffvswgmfpi lfilgpegfg 901 vlsvygstvg
htiidlmskn cwgllghylr vlihehilih gdirkttkln iggteievet 961
lvedeaeaga vnkgtgkyas resflvmrdk mkekgidvrc snakavetdv
[0055] A single mutant Chop2 of the invention may be encoded by the
following Synthetic construct hVChR1-mKate-betahChR2(L132C) gene
sequence (GenBank Accession No. JN836745, and SEQ ID NO: 11) with
the following annotations, GFP sequence is in bold, L132C Chop2
sequence is underlined:
TABLE-US-00011 1 atggattacc ctgtggcccg gtccctgatt gtaagatacc
ccaccgatct gggcaatgga 61 accgtgtgca tgcccagagg acaatgctac
tgcgaggggt ggctgaggag ccggggcact 121 agtatcgaaa aaaccatcgc
tatcaccctc cagtgggtag tgttcgctct gtccgtagcc 181 tgtctcggct
ggtatgcata ccaagcctgg agggctacct gtgggtggga ggaagtatac 241
gtggccctga tcgagatgat gaagtccatc atcgaggctt tccatgagtt cgactcccca
301 gccacactct ggctcagcag tgggaatggc gtagtgtgga tgagatatgg
agagtggctg 361 ctgacctgtc ccgtcctgct cattcatctg tccaatctga
ccgggctgaa agatgactac 421 tccaagagaa caatgggact gctggtgagt
gacgtggggt gtattgtgtg gggagccacc 481 tccgccatgt gcactggatg
gaccaagatc ctctttttcc tgatttccct ctcctatggg 541 atgtatacat
acttccacgc cgctaaggtg tatattgagg ccttccacac tgtacctaaa 601
ggcatctgta gggagctcgt gcgggtgatg gcatggacct tctttgtggc ctgggggatg
661 ttccccgtgc tgttcctcct cggcactgag ggatttggcc acattagtcc
ttacgggtcc 721 gcaattggac actccatcct ggatctgatt gccaagaata
tgtggggggt gctgggaaat 781 tatctgcggg taaagatcca cgagcatatc
ctgctgtatg gcgatatcag aaagaagcag 841 aaaatcacca ttgctggaca
ggaaatggag gtggagacac tggtagcaga ggaggaggac 901 gggaccgcgg
tcgccaccat ggtgtctaag ggcgaagagc tgattaagga gaacatgcac 961
atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc
1021 gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
gggcggccct 1081 ctccccttcg ccttcgacat cctggctacc agcttcatgt
acggcagcaa aaccttcatc 1141 aaccacaccc agggcatccc cgacttcttt
aagcagtcct tccctgaggg cttcacatgg 1201 gagagagtca ccacatacga
agacgggggc gtgctgaccg ctacccagga caccagcctc 1261 caggacggct
gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc 1321
cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagatgct gtaccccgct
1381 gacggcggcc tggaaggcag agccgacatg gccctgaagc tcgtgggcgg
gggccacctg 1441 atctgcaact tgaagaccac atacagatcc aagaaacccg
ctaagaacct caagatgccc 1501 ggcgtctact atgtggacag aagactggaa
agaatcaagg aggccgacaa agagacctac 1561 gtcgagcagc acgaggtggc
tgtggccaga tactgcgacc tccctagcaa actggggcac 1621 aaacttaatt
gcctgcagga gaagaagtca tgcagccagc gcatggccga attccggcaa 1681
tactgttgga acccggacac tgggcagatg ctgggccgca ccccagcccg gtgggtgtgg
1741 atcagcctgt actatgcagc tttctacgtg gtcatgactg ggctctttgc
cttgtgcatc 1801 tatgtgctga tgcagaccat tgatccctac acccccgact
accaggacca gttaaagtca 1861 ccgggggtaa ccttgagacc ggatgtgtat
ggggaaagag ggctgcagat ttcctacaac 1921 atctctgaaa acagctctag
acaggcccag atcaccggac gtccggagac tgagacattg 1981 ccaccggtgg
actacggggg ggccctgagc gctgtgggca gagaactcct gttcgtgaca 2041
aatccagtcg tggtgaacgg ctccgtactc gtacccgagg atcagtgcta ttgcgcagga
2101 tggatcgaga gcagaggcac aaacggcgca cagactgcat ccaacgtgct
ccagtggttg 2161 gccgcaggct tttccattct cctgctcatg ttttacgcct
accagacttg gaagtccaca 2221 tgtggctggg aggaaatcta cgtgtgtgca
atcgaaatgg tgaaggtgat cctggagttt 2281 ttcttcgaat ttaaaaaccc
aagcatgctg tacctggcta ctggccacag agtgcagtgg 2341 ctgcggtatg
ccgaatggct gctgacttgc ccagtgattc tgatccacct gtccaacctg 2401
actgggctgt ctaacgatta cagtaggaga acaatgggac tgctcgtatc cgacatcggc
2461 actatcgtat ggggcgcaac tagtgccatg gccactggat acgtgaaagt
gatcttcttc 2521 tgcctgggac tctgctacgg agcaaacaca ttttttcatg
ccgcaaaagc atatatcgag 2581 gggtatcata ccgtcccaaa gggccggtgt
agacaagtgg tgactggcat ggcttggctg 2641 ttcttcgtgt cctgggggat
gtttcccatc ctctttatcc tgggcccaga aggcttcggg 2701 gtgctgagtg
tgtatggcag taccgtagga cacactatca ttgacctgat gagcaaaaac 2761
tgctgggggc tgctcggcca ctacctgaga gtactcatcc acgagcatat cctgattcat
2821 ggcgatatcc ggaaaactac caagctcaat atcgggggca ccgagattga
agtggagaca 2881 ctcgtggagg acgaggccga ggccggagca gtgaacaaag
gcactggcaa gtatgcctcc 2941 agagaatcct ttctggtgat gcgggacaaa
atgaaggaga aaggcattga tgtacggtgc 3001 agtaatgcca aagccgtcga
gactgatgtg tag
[0056] A single mutant Chop2 of the invention may be encoded by the
following Synthetic construct hVChR1-mKate-betahChR2(L132C) amino
acid sequence (GenBank Accession No. AER29838, and SEQ ID NO: 12)
with the following annotations, GFP sequence is in bold, L132C
Chop2 sequence is underlined:
TABLE-US-00012 1 mdypvarsli vryptdlgng tvcmprgqcy cegwlrsrgt
siektiaitl qwvvfalsva 61 clgwyayqaw ratcgweevy valiemmksi
ieafhefdsp atlwlssgng vvwmrygewl 121 ltcpvllihl snitglkddy
skrtmgllvs dvgcivwgat samctgwtki lfflislsyg 181 mytyfhaakv
yieafhtvpk gicrelvrvm awtffvawgm fpvlfllgte gfghispygs 241
aighsildli aknmwgvlgn ylrvkihehi llygdirkkq kitiagqeme vetivaeeed
301 gtavatmvsk geelikenmh mklymegtvn nhhfkctseg egkpyegtqt
mrikvveggp 361 lpfafdllat sfmygsktfl nhtqgipdff kqsfpegftw
ervttyedgg vltatqdtsl 421 qdgcliynvk irgvnfpsng pvmqkktlgw
eastemlypa dgglegradm alklvggghl 481 icnlkttyrs kkpaknlkmp
gvyyvdrrle rikeadkety veqhevavar ycdlpsklgh 541 klnclqekks
csqrmaefrq ycwnpdtgqm lgrtparwvw islyyaafyv vmtglfalci 601
yvlmqtidpy tpdyqdqlks pgvtlrpdvy gerglqisyn isenssrqaq itgrpetetl
661 ppvdyggals avgrellfvt npvvvngsvl vpedqcycag wiesrgtnga
qtasnvlqwl 721 aagfsilllm fyayqtwkst cgweeiyvca iemvkvilef
ffefknpsml ylatghrvqw 781 lryaewlltc pvilihlsnl tglsndysrr
tmgllvsdig tivwgatsam atgyvkviff 841 clglcygant ffhaakayie
gyhtvpkgrc rqvvtgmawl ffvswgmfpi lfilgpegfg 901 vlsvygstvg
htiidlmskn cwgllghylr vlihehilih gdirkttkln iggteievet 961
lvedeaeaga vnkgtgkyas resflvmrdk mkekgidvrc snakavetdv
[0057] A L132C single mutant Chop2 of the invention may be encoded
by the following amino acid sequence (positions 132 underlined and
bolded, SEQ ID NO: 13):
TABLE-US-00013 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ICIHLSNLTG
LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0058] A T159C single mutant Chop2 of the invention may be encoded
by the following amino acid sequence (positions 159 underlined and
bolded, SEQ ID NO: 14):
TABLE-US-00014 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ILIHLSNLTG
LSNDYSRRTM GLLVSDIGCI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0059] A L132C/T159C double mutant Chop2 of the invention may be
encoded by the following nucleotide sequence (SEQ ID NO: 15):
TABLE-US-00015 1 atggactacg ggggggctct gtctgctgtc gggagggaac
tgctgtttgt gactaaccct 61 gtcgtcgtga acgggagtgt gctggtccct
gaggaccagt gctactgtgc cggctggatc 121 gaatcacgcg gaaccaacgg
ggcccagaca gctagcaatg tgctgcagtg gctggccgct 181 gggtttagta
tcctgctgct gatgttctac gcctatcaga cttggaagtc aacctgcggc 241
tgggaggaaa tctacgtgtg cgctattgag atggtgaaag tgatcctgga gttcttcttc
301 gagttcaaga acccaagcat gctgtacctg gctactggac accgagtgca
gtggctgaga 361 tatgcagaat ggctgctgac atgccccgtc atctgcattc
acctgtccaa cctgacaggc 421 ctgagcaatg actactccag gagaactatg
ggactgctgg tgtccgacat cggctgcatt 481 gtctggggag caacttctgc
tatggcaacc ggatacgtga aggtcatctt tttctgcctg 541 gggctgtgct
atggcgcaaa tacctttttc cacgcagcca aggcctacat tgaggggtat 601
cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg gctgtttttc
661 gtctcttggg gaatgtttcc catcctgttc attctggggc ctgaagggtt
cggcgtgctg 721 tctgtctacg gaagtacagt ggggcatact atcattgacc
tgatgtccaa aaactgttgg 781 ggcctgctgg gacactatct gagagtgctg
atccacgagc atatcctgat tcatggcgat 841 attcggaaga ccacaaaact
gaatatcggc ggaaccgaga ttgaagtgga aacactggtg 901 gaagacgagg
ctgaggctgg ggctgtgaac aaggggactg gcaaa
[0060] A L132C/T159C double mutant Chop2 of the invention may be
encoded by the following amino acid sequence (positions 132 and 159
underlined and bolded, SEQ ID NO: 16):
TABLE-US-00016 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ICIHLSNLTG
LSNDYSRRTM GLLVSDIGCI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0061] A T159S single mutant Chop2 of the invention may be encoded
by the following amino acid sequence (positions 159 underlined and
bolded, SEQ ID NO: 17):
TABLE-US-00017 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ILIHLSNLTG
LSNDYSRRTM GLLVSDIGSI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0062] A L132C/T159S double mutant Chop2 of the invention may be
encoded by the following nucleotide sequence (SEQ ID NO: 18):
TABLE-US-00018 1 atggactacg ggggggctct gtctgctgtc gggagggaac
tgctgtttgt gactaaccct 61 gtcgtcgtga acgggagtgt gctggtccct
gaggaccagt gctactgtgc cggctggatc 121 gaatcacgcg gaaccaacgg
ggcccagaca gctagcaatg tgctgcagtg gctggccgct 181 gggtttagta
tcctgctgct gatgttctac gcctatcaga cttggaagtc aacctgcggc 241
tgggaggaaa tctacgtgtg cgctattgag atggtgaaag tgatcctgga gttcttcttc
301 gagttcaaga acccaagcat gctgtacctg gctactggac accgagtgca
gtggctgaga 361 tatgcagaat ggctgctgac atgccccgtc atctgcattc
acctgtccaa cctgacaggc 421 ctgagcaatg actactccag gagaactatg
ggactgctgg tgtccgacat cggcagcatt 481 gtctggggag caacttctgc
tatggcaacc ggatacgtga aggtcatctt tttctgcctg 541 gggctgtgct
atggcgcaaa tacctttttc cacgcagcca aggcctacat tgaggggtat 601
cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg gctgtttttc
661 gtctcttggg gaatgtttcc catcctgttc attctggggc ctgaagggtt
cggcgtgctg 721 tctgtctacg gaagtacagt ggggcatact atcattgacc
tgatgtccaa aaactgttgg 781 ggcctgctgg gacactatct gagagtgctg
atccacgagc atatcctgat tcatggcgat 841 attcggaaga ccacaaaact
gaatatcggc ggaaccgaga ttgaagtgga aacactggtg 901 gaagacgagg
ctgaggctgg ggctgtgaac aaggggactg gcaaa
[0063] A L132C/T159S double mutant Chop2 of the invention may be
encoded by the following amino acid sequence (positions 132 and 159
underlined and bolded, SEQ ID NO: 19):
TABLE-US-00019 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ICIHLSNLTG
LSNDYSRRTM GLLVSDIGSI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0064] A L132A single mutant Chop2 of the invention may be encoded
by the following amino acid sequence (position 132 underlined and
bolded, SEQ ID NO: 20):
TABLE-US-00020 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV IAIHLSNLTG
LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0065] A L132A/T159C double mutant Chop2 of the invention may be
encoded by the following nucleotide sequence (SEQ ID NO: 21):
TABLE-US-00021 1 ATGGACTACG GGGGGGCTCT GTCTGCTGTC GGGAGGGAAC
TGCTGTTTGT GACTAACCCT 61 GTCGTCGTGA ACGGGAGTGT GCTGGTCCCT
GAGGACCAGT GCTACTGTGC CGGCTGGATC 121 GAATCACGCG GAACCAACGG
GGCCCAGACA GCTAGCAATG TGCTGCAGTG GCTGGCCGCT 181 GGGTTTAGTA
TCCTGCTGCT GATGTTCTAC GCCTATCAGA CTTGGAAGTC AACCTGCGGC 241
TGGGAGGAAA TCTACGTGTG CGCTATTGAG ATGGTGAAAG TGATCCTGGA GTTCTTCTTC
301 GAGTTCAAGA ACCCAAGCAT GCTGTACCTG GCTACTGGAC ACCGAGTGCA
GTGGCTGAGA 361 TATGCAGAAT GGCTGCTGAC ATGCCCCGTC ATCGCCATTC
ACCTGTCCAA CCTGACAGGC 421 CTGAGCAATG ACTACTCCAG GAGAACTATG
GGACTGCTGG TGTCCGACAT CGGCTGCATT 481 GTCTGGGGAG CAACTTCTGC
TATGGCAACC GGATACGTGA AGGTCATCTT TTTCTGCCTG 541 GGGCTGTGCT
ATGGCGCAAA TACCTTTTTC CACGCAGCCA AGGCCTACAT TGAGGGGTAT 601
CATACCGTGC CAAAAGGCCG GTGCCGACAG GTGGTCACAG GAATGGCTTG GCTGTTTTTC
661 GTCTCTTGGG GAATGTTTCC CATCCTGTTC ATTCTGGGGC CTGAAGGGTT
CGGCGTGCTG 721 TCTGTCTACG GAAGTACAGT GGGGCATACT ATCATTGACC
TGATGTCCAA AAACTGTTGG 781 GGCCTGCTGG GACACTATCT GAGAGTGCTG
ATCCACGAGC ATATCCTGAT TCATGGCGAT 841 ATTCGGAAGA CCACAAAACT
GAATATCGGC GGAACCGAGA TTGAAGTGGA AACACTGGTG 901 GAAGACGAGG
CTGAGGCTGG GGCTGTGAAC AAGGGGACTG GCAAA
[0066] A L132A/T159C double mutant Chop2 of the invention may be
encoded by the following amino acid sequence (positions 132 and 159
underlined and bolded, SEQ ID NO: 22):
TABLE-US-00022 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV IAIHLSNLTG
LSNDYSRRTM GLLVSDIGCI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0067] A T159A single mutant Chop2 of the invention may be encoded
by the following amino acid sequence (position 159 underlined and
bolded, SEQ ID NO: 23):
TABLE-US-00023 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ILIHLSNLTG
LSNDYSRRTM GLLVSDIGAI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0068] A L132C/T159A double mutant Chop2 of the invention may be
encoded by the following nucleotide sequence (SEQ ID NO: 24):
TABLE-US-00024 1 atggactacg ggggggctct gtctgctgtc gggagggaac
tgctgtttgt gactaaccct 61 gtcgtcgtga acgggagtgt gctggtccct
gaggaccagt gctactgtgc cggctggatc 121 gaatcacgcg gaaccaacgg
ggcccagaca gctagcaatg tgctgcagtg gctggccgct 181 gggtttagta
tcctgctgct gatgttctac gcctatcaga cttggaagtc aacctgcggc 241
tgggaggaaa tctacgtgtg cgctattgag atggtgaaag tgatcctgga gttcttcttc
301 gagttcaaga acccaagcat gctgtacctg gctactggac accgagtgca
gtggctgaga 361 tatgcagaat ggctgctgac atgccccgtc atctgcattc
acctgtccaa cctgacaggc 421 ctgagcaatg actactccag gagaactatg
ggactgctgg tgtccgacat cggcgccatt 481 gtctggggag caacttctgc
tatggcaacc ggatacgtga aggtcatctt tttctgcctg 541 gggctgtgct
atggcgcaaa tacctttttc cacgcagcca aggcctacat tgaggggtat 601
cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg gctgtttttc
661 gtctcttggg gaatgtttcc catcctgttc attctggggc ctgaagggtt
cggcgtgctg 721 tctgtctacg gaagtacagt ggggcatact atcattgacc
tgatgtccaa aaactgttgg 781 ggcctgctgg gacactatct gagagtgctg
atccacgagc atatcctgat tcatggcgat 841 attcggaaga ccacaaaact
gaatatcggc ggaaccgaga ttgaagtgga aacactggtg 901 gaagacgagg
ctgaggctgg ggctgtgaac aaggggactg gcaaa
[0069] A L132C/T159A double mutant Chop2 of the invention may be
encoded by the following amino acid sequence (positions 132 and 159
underlined and bolded, SEQ ID NO: 25):
TABLE-US-00025 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ICIHLSNLTG
LSNDYSRRTM GLLVSDIGAI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0070] A wild type (WT) Chop2 of the invention may be encoded by
the following amino acid sequence (SEQ ID NO: 26):
TABLE-US-00026 1 MDYGGALSAV GRELLFVTNP VVVNGSVLVP EDQCYCAGWI
ESRGTNGAQT ASNVLQWLAA 61 GFSILLLMFY AYQTWKSTCG WEEIYVCAIE
MVKVILEFFF EFKNPSMLYL ATGHRVQWLR 121 YAEWLLTCPV ILIHLSNLTG
LSNDYSRRTM GLLVSDIGTI VWGATSAMAT GYVKVIFFCL 181 GLCYGANTFF
HAAKAYIEGY HTVPKGRCRQ VVTGMAWLFF VSWGMFPILF ILGPEGFGVL 241
SVYGSTVGHT IIDLMSKNCW GLLGHYLRVL IHEHILIHGD IRKTTKLNIG GTEIEVETLV
301 EDEAEAGAVN KGTGK
[0071] Mutant ChR2 proteins of the invention also demonstrate
slower channel kinetics. Higher light sensitivity was found to
correlate with slower channel kinetics, indicating a trade-off
between light sensitivity and channel kinetics. Chop2 proteins that
form the ChR2 proteins of the present invention may also comprise
additional mutations or modifications that may improve channel
kinetics, or increase the deactivation rate, of the ChR2.
Particularly preferred ChR2 mutants balance the threshold of light
sensitivity with channel kinetics.
Compositions and Kits
[0072] Compositions and kits of the invention comprise at least one
nucleic acid molecule or polypeptide molecule that encodes a mutant
Chop2 protein, and the resulting ChR2, of the invention. The at
least one nucleic acid molecule or polypeptide molecule that
encodes a mutant Chop2 protein of the invention may further include
a pharmaceutically-acceptable carrier. Kits of the invention
further include instructions for administering a composition of the
invention to a subject.
Therapeutic Uses
[0073] Mutations were made on a codon optimized Chop2-GFP fusion
protein to create single and double mutations at the L132 (Leucine
132) and T159 (threonine 159) sites. The functional properties of
each mutant ChR2, or a combination thereof, were first examined in
HEK cells. AAV2 virus vectors carrying mutant Chop2-GFP constructs
driven by CAG promoter were made and injected intravitreally into
the eyes of adult mice. Mutant Chop2-mediated light responses were
examined by using multi-electrode array recordings from whole-mount
retinas.
[0074] Single mutant ChR2, i.e., L132 and T159C, markedly lower the
threshold light intensity that is required to evoke a ChR2-mediated
photocurrent. Moreover, several double mutant ChR2 variants,
including L132C/T159C, L132A/T159C, and L132C/T159S, were found to
further increase the photocurrent above the results of any single
mutant ChR2 at low light intensities. The double mutants exhibited
a slower off-rate, which is likely to contribute to the increased
photocurrent at the low light intensities. Spiking activity of
retinal ganglion cells mediated by the L132C/T159C double mutant
was observed at the light intensity of 10.sup.13 photon/cm.sup.2/s
and at the wavelength of 473 nm. This light level is about 1.5 to 2
log units lower than the light level that is required to elicit the
spiking activity with wild-type ChR2. The spike firing of retinal
ganglion cells expressing L132C/T159C could follow a light flicker
frequency of up to 15 Hz. Ongoing studies are evaluating the
long-term expression and safety of mutant ChR2s of the invention in
retinal neurons.
[0075] Furthermore, expression of the mutant Chop2 proteins, and
the resulting ChR2 proteins, of the present invention was not found
to cause neurotoxicity of up to two months after viral injection in
mice, demonstrating the safety of the present invention for
therapeutic use.
[0076] Vectors for use in the present invention can include various
viral vectors, such as plasmids and recombinant viruses, i.e.,
recombinant adeno-associated virus (rAAV), recombinant
adenoviruses, recombinant retroviruses, recombinant lentiviruses,
and other viruses known in the art.
[0077] In some embodiments, the expression of the Chop2 proteins of
the present invention is driven by a constitutive promoter, i.e.,
CAG promoter, CMV promoter, LTR. In other embodiments, the promoter
is an inducible or a cell-specific promoter. Cell type-specific
promoters that enable Chop2 protein expression in specific
subpopulations of cells, i.e., retinal neuron cells or degenerating
cells, may be preferred. These cells may include, but are not
limited to, a retinal ganglion cell, a photoreceptor cell, a
bipolar cell, a rod bipolar cell, an ON-type cone bipolar cell, a
retinal ganglion cell, a photosensitive retinal ganglion cell, a
horizontal cell, an amacrine cell, or an AII amacrine cell. Cell
type-specific promoters are well known in the art. Particularly
preferred cell type-specific promoters include, but are not limited
to mGluR6, NK-3, and Pcp2(L7).
[0078] In some embodiments, use of different opsin genes in
addition to the mutant Chop2 proteins of the present invention and
targeted gene expression may further increase light sensitivity or
improve vision. Visual information is processed through the retina
through two pathways: an ON pathway which signals the light ON, and
an OFF pathway which signals the light OFF. The existence of the ON
and OFF pathway is important for the enhancement of contrast
sensitivity. The visual signal in the ON pathway is relay from
ON-cone bipolar cells to ON ganglion cells. Both ON-cone bipolar
cells and ON-ganglion cells are depolarized in response to light.
On the other hand, the visual signal in the OFF pathway is carried
from OFF-cone bipolar cells to OFF ganglion cells. Both OFF-cone
bipolar cells and OFF-ganglion cells are hypopolarized in response
to light. Rod bipolar cells, which are responsible for the ability
to see in dim light (scotopic vision), are ON bipolar cells
(depolarized in response to light). Rod bipolar cells relay the
vision signal through AII amacrine cells (an ON type retinal cells)
to ON an OFF cone bipolar cells.
[0079] Accordingly, a dual rhodopsin system can be used to
recapitulate the ON and OFF pathways integral to visual processing
and acuity. Briefly, a Chop2 protein of the present invention can
be specifically targeted to ON type retinal neurons (i.e., ON type
ganglion cells and/or ON type bipolar cells), while a
hypopolarizing light sensor (i.e., halorhodopsin or other chloride
pump known in the art) can be targeted to OFF type retinal neurons
(i.e. OFF type ganglion cells and/or OFF type bipolar cells) to
create ON and OFF pathways. The specific targeting to preferred
cell subpopulations can be achieved through the use of different
cell type-specific promoters. For example, Chop2 expression may be
driven by the mGluR6 promoter for targeted expression in ON-type
retinal neurons (i.e., ON type ganglion cells and/or ON type
bipolar cells) while a hypopolarizing channel, such as
halorhodopsin, expression is driven by the NK-3 promoter for
targeted expression in OFF-type retinal neurons (i.e., OFF type
ganglion cells and/or OFF type bipolar cells).
[0080] An alternative approach to restore ON and OFF pathways in
the retina is achieved by, expressing a depolarizing light sensor,
such as ChR2, to rod bipolar cells or AII amacrine. In this
approach, the depolarization of rod bipolar cells or AII amacrine
cells can lead to the ON and OFF responses at the levels of cone
bipolar cells and the downstream retinal ganglion cells. Thus, the
ON and OFF pathways that are inherent in the retina are
maintained.
[0081] The present invention can be formulated to a pharmaceutical
composition or medicament suitable for administration into a
subject or patient. Suitable routes of administration include, for
example, intravitreal, intraocular, or subretinal injection.
[0082] Such formulations comprise a pharmaceutically and/or
physiologically acceptable vehicle, diluent, carrier or excipient,
such as buffered saline or other buffers, e.g., HEPES, to maintain
physiologic pH. For a discussion of such components and their
formulation, see, generally, Gennaro, A E., Remington: The Science
and Practice of Pharmacy, Lippincott Williams & Wilkins
Publishers; 2003 or latest edition). See also, WO00/15822. If the
preparation is to be stored for long periods, it may be frozen, for
example, in the presence of glycerol.
[0083] The pharmaceutical composition described above is
administered to a subject having a visual or blinding disease by
any appropriate route, preferably by intravitreal or subretinal
injection, depending on the retinal layer being targeted.
[0084] Disclosures from Bennett and colleagues (cited herein)
concern targeting of retinal pigment epithelium--the most distal
layer from the vitreal space. According to the present invention,
the Chop2 construct or polypeptide is targeted to retinal cells,
i.e., retinal ganglion cells or bipolar cells. Such cells are known
to be reasonably well-accessible to intravitreal injection as
disclosed herein. Intravitreal and/or subretinal injection can
provide the necessary access to the bipolar cells, especially in
circumstances in which the photoreceptor cell layer is absent due
to degeneration--which is the case in certain forms of degeneration
that the present invention is intended to overcome.
[0085] To test for the vector's ability to express the Chop2
mutants of the present invention, specifically in mammalian retinal
neurons, by AAV-mediated delivery, a combination of a preferred
promoter sequence linked to a reporter gene such as LacZ or GFP
linked to a SV40 poly A sequence can be inserted into a plasmid and
packaged into rAAV virus particles, concentrated, tested for
contaminating adenovirus and titered for rAAV using an infectious
center assay. The right eyes of a number of test subjects,
preferably inbred mice, can be injected sub-retinally with about 1
.mu.l of the rAAV preparation (e.g., greater than about 10.sup.10
infectious units ml). Two weeks later, the right (test) and left
(control) eyes of half the animals may be removed, fixed and
stained with an appropriate substrate or antibody or other
substance to reveal the presence of the reporter gene. A majority
of the test retinas in injected eyes will exhibited a focal stained
region, e.g., blue for LacZ/Xgal, or green for GFP consistent with
a subretinal bleb of the injected virus creating a localized
retinal detachment. All control eyes may be negative for the
reporter gene product. Reporter gene expression examined in mice
sacrificed at later periods is detected for at least 10 weeks
post-injection, which suggests persistent expression of the
reporter transgene.
[0086] In one embodiment, the Chop2 constructs are packaged in
adenoviral vectors for transgene delivery. An effective amount of
rAAV virions carrying a nucleic acid sequence encoding the Chop2
DNA under the control of the promoter of choice, preferably a
constitutive CMV promoter or a cell-specific promoter such as
mGluR6, is preferably in the range of between about 10.sup.10 to
about 10.sup.13 rAAV infectious units in a volume of between about
150 and about 800 .mu.l per injection. The rAAV infectious units
can be measured according to McLaughlin, S K et al., 1988, J Virol
62:1963. More preferably, the effective amount is between about
10.sup.10 and about 10.sup.12 rAAV infectious units and the
injection volume is preferably between about 250 and about 500
.mu.l. Other dosages and volumes, preferably within these ranges
but possibly outside them, may be selected by the treating
professional, taking into account the physical state of the subject
(preferably a human), who is being treated, including, age, weight,
general health, and the nature and severity of the particular
ocular disorder.
[0087] It may also be desirable to administer additional doses
("boosters") of the present nucleic acid(s) or rAAV compositions.
For example, depending upon the duration of the transgene
expression within the ocular target cell, a second treatment may be
administered after 6 months or yearly, and may be similarly
repeated. Neutralizing antibodies to AAV are not expected to be
generated in view of the routes and doses used, thereby permitting
repeat treatment rounds.
[0088] The need for such additional doses can be monitored by the
treating professional using, for example, well-known
electrophysiological and other retinal and visual function tests
and visual behavior tests. The treating professional will be able
to select the appropriate tests applying routine skill in the art.
It may be desirable to inject larger volumes of the composition in
either single or multiple doses to further improve the relevant
outcome parameters.
Ocular Disorders
[0089] The ocular disorders for which the present Chop2 proteins,
and the resulting ChR2 proteins, are intended and may be used to
improve one or more parameters of vision include, but are not
limited to, developmental abnormalities that affect both anterior
and posterior segments of the eye. Anterior segment disorders
include glaucoma, cataracts, corneal dystrophy, keratoconus.
Posterior segment disorders include blinding disorders caused by
photoreceptor malfunction and/or death caused by retinal
dystrophies and degenerations. Retinal disorders include congenital
stationary night blindness, age-related macular degeneration,
congenital cone dystrophies, and a large group of
retinitis-pigmentosa (RP)-related disorders. These disorders
include genetically pre-disposed death of photoreceptor cells, rods
and cones in the retina, occurring at various ages. Among those are
severe retinopathies, such as subtypes of RP itself that progresses
with age and causes blindness in childhood and early adulthood and
RP-associated diseases, such as genetic subtypes of LCA, which
frequently results in loss of vision during childhood, as early as
the first year of life. The latter disorders are generally
characterized by severe reduction, and often complete loss of
photoreceptor cells, rods and cones. (Trabulsi, E I, ed., Genetic
Diseases of the Eye, Oxford University Press, NY, 1998).
[0090] In particular, the Chop2 and ChR2 proteins of the present
invention useful for the treatment and/or restoration of at least
partial vision to subjects that have lost vision due to ocular
disorders, such as RPE-associated retinopathies, which are
characterized by a long-term preservation of ocular tissue
structure despite loss of function and by the association between
function loss and the defect or absence of a normal gene in the
ocular cells of the subject. A variety of such ocular disorders are
known, such as childhood onset blinding diseases, retinitis
pigmentosa, macular degeneration, and diabetic retinopathy, as well
as ocular blinding diseases known in the art. It is anticipated
that these other disorders, as well as blinding disorders of
presently unknown causation which later are characterized by the
same description as above, may also be successfully treated by the
Chop2 and ChR2 proteins of the present invention. Thus, the
particular ocular disorder treated by the present invention may
include the above-mentioned disorders and a number of diseases
which have yet to be so characterized.
Optogenetics
[0091] The emerging field of optogenetics involves the combination
of genetic and optical methods to control specific events in
targeted cells of a living tissue. Optogenetics may be used within
freely moving mammals and other animals. Moreover, the temporal
precision (millisecond-timescale) of optogeneic methods are
sufficient to function within intact biological systems.
[0092] The instant invention provides Chop2-gene therapy to retinal
tissues of the eye, by introducing into retinal cells a nucleic
acid or polypeptide encoding for at least one mutant form of Chop2.
Mutant Chop2/ChR2 proteins of the invention are specifically
adapted to be light-activated at lower thresholds of light
intensities than their wild type counterparts. Accordingly, the
mutant Chop2/ChR2 proteins of the invention can be used to activate
cells of the retina and visual system using less damaging sources
of illumination. The mutant Chop2/ChR2 proteins also conduct larger
photocurrents upon activation, resulting in a more robust or
efficacious response from the mutant Chop2/ChR2-expressing
cells.
[0093] For example, mutant Chop2 proteins of the invention are
administered to a subject through local, intravitreous or
subretinal, injection of a nucleic acid molecule encoding a mutant
Chop2, a mutant Chop2 polypeptide molecule, or a cell expressing a
mutant Chop2/ChR2. Retinal cells of the subject express the mutant
Chop2 proteins within the plasma membrane. When the transfected or
transformed retinal cells encounter light radiation, the
transfected or transformed retinal cells transduce an improved or
restored signal.
[0094] These methods may be used in subjects of normal and/or
impaired vision. Chop2/ChR2 mutants of the invention may preserve,
improve, or restore vision. Moreover, Chop2/ChR2 mutants of the
invention are used to preserve, improve, or restore the
transduction of non-visual information from photosensitive retinal
ganglion cells to the brain.
[0095] The term "vision" as used herein is defined as the ability
of an organism to usefully detect light as a stimulus for
differentiation or action. Vision is intended to encompass the
following: [0096] 1. Light detection or perception--the ability to
discern whether or not light is present; [0097] 2. Light
projection--the ability to discern the direction from which a light
stimulus is coming; [0098] 3. Resolution--the ability to detect
differing brightness levels (i.e., contrast) in a grating or letter
target; and [0099] 4. Recognition--the ability to recognize the
shape of a visual target by reference to the differing contrast
levels within the target. Thus, "vision" includes the ability to
simply detect the presence of light. The polypeptides and
polynucleotides encoding mutant Chop2 of the present invention can
be used to improve or restore vision, wherein the improvement or
restoration in vision includes, for example, increases in light
detection or perception, increase in light sensitivity or
photosensitivity in response to a light stimulus, increase in the
ability to discern the direction from which a light stimulus is
coming, increase in the ability to detect differing brightness
levels, increase in the ability to recognize the shape of a visual
target, and increases in visual evoked potential or transmission
from the retina to the cortex. As such, improvement or restoration
of vision may or may not include full restoration of sight, i.e.,
wherein the vision of the patient treated with the present
invention is restored to the degree to the vision of a non-affected
individual. The visual recovery described in the animal studies
described below may, in human terms, place the person on the low
end of vision function by increasing one aspect of vision (i.e.,
light sensitivity, or visual evoked potential) without restoring
full sight. Nevertheless, placement at such a level would be a
significant benefit because these individuals could be trained in
mobility and potentially in low order resolution tasks which would
provide them with a greatly improved level of visual independence
compared to total blindness. Even basic light perception can be
used by visually impaired individuals, whose vision is improved
using the present compositions and methods, to accomplish specific
daily tasks and improve general mobility, capability, and quality
of life.
[0100] The degree of restoration of vision can be determined
through the measurement of vision before, and preferably after,
administering a vector comprising, for example, DNA encoding Chop2.
Vision can be measured using any of a number of methods well-known
in the art or methods not yet established. Vision, as improved or
restored by the present invention, can be measured by any of the
following visual responses: [0101] 1. a light detection response by
the subject after exposure to a light stimulus--in which evidence
is sought for a reliable response of an indication or movement in
the general direction of the light by the subject individual when
the light it is turned on; [0102] 2. a light projection response by
the subject after exposure to a light stimulus in which evidence is
sought for a reliable response of indication or movement in the
specific direction of the light by the individual when the light is
turned on; [0103] 3. light resolution by the subject of a light vs.
dark patterned visual stimulus, which measures the subject's
capability of resolving light vs dark patterned visual stimuli as
evidenced by: [0104] a. the presence of demonstrable reliable
optokinetically produced nystagmoid eye movements and/or related
head or body movements that demonstrate tracking of the target (see
above) and/or [0105] b. the presence of a reliable ability to
discriminate a pattern visual stimulus and to indicate such
discrimination by verbal or non-verbal means, including, for
example pointing, or pressing a bar or a button; or [0106] 4.
electrical recording of a visual cortex response to a light flash
stimulus or a pattern visual stimulus, which is an endpoint of
electrical transmission from a restored retina to the visual
cortex, also referred to as the visual evoked potential (VEP).
Measurement may be by electrical recording on the scalp surface at
the region of the visual cortex, on the cortical surface, and/or
recording within cells of the visual cortex.
[0107] Thus, improvement or restoration of vision, according to the
present invention, can include, but is not limited to: increases in
amplitude or kinetics of photocurrents or electrical response in
response to light stimulus in the retinal cells, increases in light
sensitivity (i.e., lowering the threshold light intensity required
for initiating a photocurrent or electrical response in response to
light stimulus, thereby requiring less or lower light to evoke a
photocurrent) of the retinal cells, increases in number or
amplitude of light-evoked spiking or spike firings, increases in
light responses to the visual cortex, which includes increasing in
visual evoked potential transmitted from the retina or retinal
cells to the visual cortex or the brain.
[0108] Both in vitro and in vivo studies to assess the various
parameters of the present invention may be used, including
recognized animal models of blinding human ocular disorders. Large
animal models of human retinopathy, e.g., childhood blindness, are
useful. The examples provided herein allow one of skill in the art
to readily anticipate that this method may be similarly used in
treating a range of retinal diseases.
[0109] While earlier studies by others have demonstrated that
retinal degeneration can be retarded by gene therapy techniques,
the present invention demonstrates a definite physiological
recovery of function, which is expected to generate or improve
various parameters of vision, including behavioral parameters.
[0110] Behavioral measures can be obtained using known animal
models and tests, for example performance in a water maze, wherein
a subject in whom vision has been preserved or restored to varying
extents will swim toward light (Hayes, J M et al., 1993, Behav
Genet 23:395-403).
[0111] In models in which blindness is induced during adult life or
congenital blindness develops slowly enough that the individual
experiences vision before losing it, training of the subject in
various tests may be done. In this way, when these tests are
re-administered after visual loss to test the efficacy of the
present compositions and methods for their vision-restorative
effects, animals do not have to learn the tasks de novo while in a
blind state. Other behavioral tests do not require learning and
rely on the instinctiveness of certain behaviors. An example is the
optokinetic nystagmus test (Balkema G W et al., 1984, Invest
Ophthalmol Vis Sci. 25:795-800; Mitchiner J C et al., 1976, Vision
Res. 16:1169-71).
[0112] The present invention may also be used in combination with
other forms of vision therapy known in the art to improve or
restore vision. For example, the use of visual prostheses, which
include retinal implants, cortical implants, lateral geniculate
nucleus implants, or optic nerve implants. Thus, in addition to
genetic modification of surviving retinal neurons using the present
methods, the subject being treated may be provided with a visual
prosthesis before, at the same time as, or after the molecular
method is employed. The effectiveness of visual prosthetics can be
improved with training of the individual, thus enhancing the
potential impact of the Chop2 transformation of patient cells as
contemplated herein. Training methods, such as habituation training
characterized by training the subject to recognize (i) varying
levels of light and/or pattern stimulation, and/or (ii)
environmental stimulation from a common light source or object as
would be understood by one skilled in the art; and orientation and
mobility training characterized by training the subject to detect
visually local objects and move among said objects more effectively
than without the training. In fact, any visual stimulation
techniques that are typically used in the field of low vision
rehabilitation are applicable here.
EXAMPLES
Example 1: Generation of Labeled Mutant Chop2 Constructs
[0113] Mutations were made on a codon optimized Chop2-GFP fusion
protein to create single and double mutations at the L132 (Leucine
132) and T159 (Threonine 159) sites. Several mutants were
generated, for example, single mutants such as L132A, L132C, T159A,
T159C, and T 159S, and double mutants such as L132C/T159C,
L132C/T159S, L132A/T159C, and L132C/T159A. Chop2-GFP transgenes
were cloned into a rAAV vector under the control of a CAG promoter
using methods known in the art.
Example 2: In Vitro Analysis of Mutant Chop2 Constructs
[0114] The functional properties of each mutant Chop2, or a
combination thereof, were first examined in HEK cells. Chop2
constructs were delivered to HEK cells by adenoviral infection, for
example. Upon expression of the WT or mutant Chop2, functional WT
and mutant ChR2 channels were formed. Measurements of the light
sensitivity and other properties of the ChR2 channels were assessed
as described herein. The light stimuli (photons/cm.sup.2s at 460
nm) were generated by a xenon arc lamp and attenuated by neutral
density filters: ND4.0 (2.8.times.10.sup.14), ND3.0
(1.4.times.10.sup.15), ND2.5 (4.8.times.10.sup.15); ND2.0
(1.6.times.10.sup.16), ND1.0 (1.3.times.10.sup.17), ND0
(1.2.times.10.sup.18). Light evoked currents were measured from
wild-type ChR2, T159C, L132C, L132C/T159C, and L132C/T159S. Patch
clamp recordings were performed using methods known in the art.
[0115] Representative recordings from this experiment comparing
light sensitivity between the Chop2 constructs demonstrated that
mutations at L132 alone or in combination with mutation at T159
show increased photocurrent in comparison to WT (FIG. 1A). FIG. 1B
shows the same current traces at a different scale to illustrate
the difference in amplitude of the photocurrents between WT ChR2
and ChR2 mutants more clearly. FIG. 1B specifically compares the
current traces resulting from light stimulation using the neutral
density filter (ND 2.5), equivalent to 4.8.times.10.sup.15
photos/cm.sup.2/s; the traces are designated by the arrows. The
amplitude of the photocurrent of the L132C mutant is larger than
that of WT; the amplitude of the photocurrent of double mutant
L132C/T159C is larger than that of L132C; and the amplitude of the
photocurrent of the L132C/T159S mutant larger than L132/T159C. The
current traces of the ChR2 mutants, particularly double mutants
L132C/T159C and L132C/T159S, also show slower deactivation kinetics
when compared to WT and L132C.
[0116] FIG. 2 shows the representative recordings of light-evoked
currents from WT ChR2, L132C, L132C/T159C, and L132C/T159S after
stimulation by a 10 ms light pulse (1.2.times.10.sup.18
photons/cm.sup.2/s at 460 nm wavelength) to compare the
deactivation time course, or decay time course after the light is
off. Mutant ChR2 show longer deactivation time courses, with the
double mutant L132C/T159S having the longest. Higher light
sensitivity, as demonstrated by L132C/T159C and L132C/T159S, may be
correlated with slower channel kinetics.
Example 3: In Vivo Ocular Administration and Analysis of Mutant
Chop2 Constructs
[0117] AAV2 virus vectors carrying mutant Chop2-GFP constructs
driven by CAG promoter were made and injected intravitreally into
the eyes of C57BL/6J adult mice. Adult mice were anesthetized by IP
injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Under a
dissecting microscope, an incision was made by scissors through the
eyelid to expose the sclera. A small perforation was made in the
sclera region posterior to the lens with a needle and viral vector
suspension of 0.8-1.5 .mu.l at the concentration of approximately
10.sup.11 genomic particles/ml was injected into intravitreal space
through the hole with a Hamilton syringe with a 32-gauge
blunt-ended needle. For each animal, usually only one eye was
injected with viral vectors carrying a Chop2 construct, and the
other eye was uninjected or injected with control viral vectors
carrying GFP alone. Upon expression of the WT or mutant Chop2 of
the present invention, functional WT or mutant ChR2 channels were
formed utilizing endogenous retinal, and the properties of these
ChR2 proteins were assessed as described herein.
[0118] ChR2-mediated light responses were examined by using
multi-electrode array recordings from whole-mount retinas. Light
stimuli (photons/cm.sup.2/s) was generated by a 473 nm blue laser
and attenuated by neutral density filters: ND0
(6.3.times.10.sup.16), ND1.0 (7.4.times.10.sup.15), ND1.5
(2.7.times.10.sup.15), ND2.0 (7.3.times.10.sup.14), ND2.5
(3.2.times.10.sup.14), ND3.0 (8.5.times.10.sup.13), ND3.5
(3.8.times.10.sup.13), and ND4.0 (9.5.times.10.sup.12).
[0119] The multielectrode array recordings were based on the
procedures reported by Tian and Copenhagen (2003). Briefly, the
retina was dissected and placed photoreceptor side down on a
nitrocellulose filter paper strip (Millipore Corp., Bedford,
Mass.). The mounted retina was placed in the MEA-60 multielectrode
array recording chamber of 30 .mu.m diameter electrodes spaced 200
.mu.m apart (Multi Channel System MCS GmbH, Reutlingen, Germany),
with the ganglion cell layer facing the recording electrodes. The
retina was continuously perfused in oxygenated extracellular
solution at 34.degree. C. during all experiments. The extracellular
solution contained (in mM): NaCl, 124; KCl, 2.5; CaCl.sub.2, 2;
MgCl.sub.2, 2; NaH.sub.2PO.sub.4, 1.25; NaHCO.sub.3, 26; and
glucose, 22 (pH 7.35 with 95% O.sub.2 and 5% CO.sub.2). Recordings
were usually started 60 min after the retina was positioned in the
recording chamber. The interval between onsets of each light
stimulus was 10-15 s. The signals were filtered between 200 Hz (low
cut off) and 20 kHz (high cut off). The responses from individual
neurons were analyzed using Offline Sorter software (Plexon, Inc.,
Dallas, Tex.).
[0120] Single mutant Chop2/ChR2 mutants, i.e., L132 and T159C,
markedly lower the threshold light intensity that is required to
evoke a ChR2-mediated photocurrent. Moreover, several double
mutants, including L132C/T159C, L132A/T159C, and L132C/T159S, were
found to further increase the photocurrent at low light
intensities. Different neutral density filters were used to
attenuate the light stimuli to differentiate the light-evoked
responses of the Chop2 constructs in low light. Spiking activity of
retinal ganglion cells mediated by the mutants of the present
invention was observed at the light intensities about 1.5 to 2 log
units lower than the light level that is required to elicit the
spiking activity with wild-type ChR2 (FIG. 3). Specifically, WT
ChR2 exhibited did not exhibit any spiking activity in response to
light stimuli with neutral density filter 2.5 (3.2.times.10.sup.14
photons/cm.sup.2/s) while ChR2 mutants (L132C, L132C/T159C, and
L132C/T159S) demonstrate spiking activity. In fact, the ChR2
mutants still exhibited spiking activity in response to light with
neutral density filters 3.0 and 3.5. Therefore, ChR2 mutants of the
present invention possess higher light sensitivity and, thus, a
markedly lower threshold light intensity that is required to elicit
a ChR2-mediated photocurrent. Moreover, ChR2 double mutants possess
a higher light sensitivity than single mutants, i.e. L132C. In
addition, the spike firing of retinal ganglion cells expressing
L132C/T159C and L132/T159S could follow a light flicker frequency
of up to 15 Hz and 5 Hz, respectively (FIG. 4A-B).
[0121] The L132C/T159A mutant shows high light sensitivity,
probably the most light sensitive among these mutants, but it also
shows extremely slow off-rate (the channel continue open for many
many sends after light off). Interestingly, it can be turned off
more quickly using a light with long-wavelengths, such as yellow
light. The L132C/T159A mutant (encoded by SEQ ID NOs: 24 and 25)
demonstrates significant potential.
[0122] Given the trade-off between light sensitivity and channel
kinetics, Chop2/ChR2 mutants that demonstrate a balance between
light sensitivity and channel kinetics, such as L132C/T159C or
L132C/T159S, may be suitable for the application of vision
restoration.
Example 4: Analysis of Mutant Chop2 Constructs in Mouse Models of
Disease
[0123] Mouse models of degenerative ocular diseases are known in
the art. For example, homozygous rd1 (rd1/rd1) mice are a commonly
used photoreceptor degeneration model. Rd1 mice carry a null
mutation in a cyclic GMP phosphodiesterase, PDE6, similar to some
forms of retinitis pigmentosa in humans. Other well-established
mouse models of ocular disease that may be of particular interest
to demonstrate ChR2 mutant safety and efficacy include rds (also
known as Prph.sup.Rd2), rd3, rd4, rd5, rd6, rd7, rd8, rd9,
Pde6b.sup.rd10, or cpfl1 mice.
[0124] The Chop2-GFP constructs of the present invention can be
injected intravitreally into the eyes of newborn (P1) or adult mice
at 2-12 months of age. GFP signal can be observed in the
Chop2-GFP-injected retinas, to determine the levels of ChR2
expression or expression in particular populations of cells, such
as the retinal ganglion cells. Mutant Chop2-GFP expression can be
monitored for a predetermined amount of time, i.e. 3-6 months, or 1
year after viral injection. Patch-clamp and multichannel array
recordings can be performed using the methods known in the art and
described herein to measure the light-evoked responses of mutant
Chop2-GFP-expressing cells in vivo.
[0125] Additional techniques and tests are well-established in the
art to test for the restoration of light sensitivity or vision.
Visual evoked potentials from the Chop2-GFP expressing cells or
visual cortex can be examined, as described in PCT publication WO
2007/131180. Other tests include behavioral assessments of the
visual acuity in the mice, i.e., virtual optomotor test and visual
water maze.
Example 5: Analysis of Long-Term Expression and Safety of
Administration of Mutant Chop2 Constructs to Retinal Neurons
[0126] Neurotoxicity was assessed in C57BL/6J adult mice injected
with Chop2 constructs of the present invention. The expression
safety of Chop2 mutants in the retina was assessed by
immunostaining and cell counting after exposure to strong blue
light for two weeks. None of the mice were found to exhibit
symptoms of neurotoxicity for up to two months after injection.
[0127] Additional ongoing studies are evaluating the long-term
expression and safety of Chop2/ChR2 mutants of the invention in
retinal neurons.
Other Embodiments
[0128] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
[0129] The patent and scientific literature referred to herein
establishes the knowledge that is available to those with skill in
the art. All United States patents and published or unpublished
United States patent applications cited herein are incorporated by
reference. All published foreign patents and patent applications
cited herein are hereby incorporated by reference. All other
published references, documents, manuscripts and scientific
literature cited herein are hereby incorporated by reference.
[0130] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
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tgggcagcat gggaggcatg 2100ggcggaatga gcggaatggg aggcatgggt
ggaatggggg gcatgggcgg cgccggcgcc 2160gccacgacgc aggctgcggg
cggcaacgcg gaggcggaga tgctgcagaa tctcatgaac 2220gagatcaatc
gcctgaagcg cgagcttggc gagtaaaagg ctggaggccg gtactgcgat
2280acctgcgagc tcgcgcgcct gactcgtcgt acacacggct caggagcacg
cgcgcgtgga 2340cttctcaacc tgtgtgcaac gtatctagag cggcctgtgc
gcgaccgtcc gtgagcattc 2400cggtgcgatc ttcccgcctt cgcaccgcaa
gttcccttcc tggccctgct gcgcctgacg 2460catcgtccga acggaagggc
ggcttgatca gtaaagcatt gaagactgaa gtcgtgcgac 2520cgtagtgcta
tggctctgca cgtaagtggg cgctgccctg cttactacgc attgcccaag
2580actgcttcct tttggtggcc gaggccctgg tcccacatca ttcatttgca
taacgtactg 2640tttagttaca tacgctttgc ttaacctcga caattgcaac
atgggctgag agtccgtacg 2700gcggctatgg acgaaggtgt tatcggatgt
gattaggaat ctcggttgaa aggcttcgag 2760aaagtgagct tcatctgtgg
cttctgttgg ggtcatcaag aagaacgacg gtaaggcaaa 2820cgaggtaaaa
gtggcacgtc tttgtgcaca acgggcccgt ggagagtggg ggagtgcatg
2880tgtgcggtcc taacacgcga gtgcaaagcg ggcttttctg gagctgggtt
acggtctggc 2940tcggcaactg ctctgtgttt taaccacagc ttcggaagtc
tgggtatgtt ttgttggcag 3000aaacatttgg gtaacttgag ggtgattcgt
ctggagtcgg acaacatggc tgccgtccgt 3060gtgcagggac ggtaatcaat
gagctggagc tgtgatgctc accacacgtt gcatacccct 3120gcttacaaaa
acactttgat gtcgtggcca aactatgcgt gagcaaagag ttaaagaggc
3180atgagtgcat ggttgcggac gtgcgcaaca attgcatcaa gtatttgacg
ccttcaagcc 3240aacaagtgcg cgcgcggcaa cttgattaac acgccggacg
cagtggtggg ggcgtgtaca 3300gtgtttatga gctgccattc tgcgatccgt
agtgttaggt tgcgtgtgac gccgcgcggc 3360tgtgggccct tacatggaga
gttgggtgct tcaccacacg gttggcgccg ctgaagggtg 3420tgctatgttt
tggtaaagcc ggggccctga agaccgcaac cgtagaaccg tactgaaagg
3480gtgtcagccc ggggtaactg gatgccctgg gacatagcta ttaatgttga
agtgaagccg 3540tcaagccgag tgccgtgcgc cgctgtatca ccaaggcccg tccta
35852737PRTChlamydomonas reinhardtii 2Met Asp Tyr Gly Gly Ala Leu
Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn Pro Val Val
Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln Cys Tyr Cys Ala
Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln Thr Ala Ser
Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60Leu Leu Leu
Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75 80Trp
Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile Leu 85 90
95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala Thr
100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu Leu
Thr Cys 115 120 125Pro Val Ile Leu Ile His Leu Ser Asn Leu Thr Gly
Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met Gly Leu Leu Val
Ser Asp Ile Gly Thr Ile145 150 155 160Val Trp Gly Ala Thr Ser Ala
Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe Phe Cys Leu Gly
Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190Ala Lys Ala
Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200 205Arg
Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly 210 215
220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly Val
Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile Ile
Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu Gly His Tyr
Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile His Gly Asp
Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly Thr Glu Ile
Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu Ala Gly Ala
Val Asn Lys Gly Thr Gly Lys Tyr Ala Ser Arg Glu305 310 315 320Ser
Phe Leu Val Met Arg Asp Lys Met Lys Glu Lys Gly Ile Asp Val 325 330
335Arg Ala Ser Leu Asp Asn Ser Lys Glu Val Glu Gln Glu Gln Ala Ala
340 345 350Arg Ala Ala Met Met Met Met Asn Gly Asn Gly Met Gly Met
Gly Met 355 360 365Gly Met Asn Gly Met Asn Gly Met Gly Gly Met Asn
Gly Met Ala Gly 370 375 380Gly Ala Lys Pro Gly Leu Glu Leu Thr Pro
Gln Leu Gln Pro Gly Arg385 390 395 400Val Ile Leu Ala Val Pro Asp
Ile Ser Met Val Asp Phe Phe Arg Glu 405 410 415Gln Phe Ala Gln Leu
Ser Val Thr Tyr Glu Leu Val Pro Ala Leu Gly 420 425 430Ala Asp Asn
Thr Leu Ala Leu Val Thr Gln Ala Gln Asn Leu Gly Gly 435 440 445Val
Asp Phe Val Leu Ile His Pro Glu Phe Leu Arg Asp Arg Ser Ser 450 455
460Thr Ser Ile Leu Ser Arg Leu Arg Gly Ala Gly Gln Arg Val Ala
Ala465 470 475 480Phe Gly Trp Ala Gln Leu Gly Pro Met Arg Asp Leu
Ile Glu Ser Ala 485 490 495Asn Leu Asp Gly Trp Leu Glu Gly Pro Ser
Phe Gly Gln Gly Ile Leu 500 505 510Pro Ala His Ile Val Ala Leu Val
Ala Lys Met Gln Gln Met Arg Lys 515 520 525Met Gln Gln Met Gln Gln
Ile Gly Met Met Thr Gly Gly Met Asn Gly 530 535 540Met Gly Gly Gly
Met Gly Gly Gly Met Asn Gly Met Gly Gly Gly Asn545 550 555 560Gly
Met Asn Asn Met Gly Asn Gly Met Gly Gly Gly Met Gly Asn Gly 565 570
575Met Gly Gly Asn Gly Met Asn Gly Met Gly Gly Gly Asn Gly Met Asn
580 585 590Asn Met Gly Gly Asn Gly Met Ala Gly Asn Gly Met Gly Gly
Gly Met 595 600 605Gly Gly Asn Gly Met Gly Gly Ser Met Asn Gly Met
Ser Ser Gly Val 610 615 620Val Ala Asn Val Thr Pro Ser Ala Ala Gly
Gly Met Gly Gly Met Met625 630 635 640Asn Gly Gly Met Ala Ala Pro
Gln Ser Pro Gly Met Asn Gly Gly Arg 645 650 655Leu Gly Thr Asn Pro
Leu Phe Asn Ala Ala Pro Ser Pro Leu Ser Ser 660 665 670Gln Leu Gly
Ala Glu Ala Gly Met Gly Ser Met Gly Gly Met Gly Gly 675 680 685Met
Ser Gly Met Gly Gly Met Gly Gly Met Gly Gly Met Gly Gly Ala 690 695
700Gly Ala Ala Thr Thr Gln Ala Ala Gly Gly Asn Ala Glu Ala Glu
Met705 710 715 720Leu Gln Asn Leu Met Asn Glu Ile Asn Arg Leu Lys
Arg Glu Leu Gly 725 730 735Glu32241DNAChlamydomonas reinhardtii
3gcatctgtcg ccaagcaagc attaaacatg gattatggag gcgccctgag tgccgttggg
60cgcgagctgc tatttgtaac gaacccagta gtcgtcaatg gctctgtact tgtgcctgag
120gaccagtgtt actgcgcggg ctggattgag tcgcgtggca caaacggtgc
ccaaacggcg 180tcgaacgtgc tgcaatggct tgctgctggc ttctccatcc
tactgcttat gttttacgcc 240taccaaacat ggaagtcaac ctgcggctgg
gaggagatct atgtgtgcgc tatcgagatg 300gtcaaggtga ttctcgagtt
cttcttcgag tttaagaacc cgtccatgct gtatctagcc 360acaggccacc
gcgtccagtg gttgcgttac gccgagtggc ttctcacctg cccggtcatt
420ctcattcacc tgtcaaacct gacgggcttg tccaacgact acagcaggcg
caccatgggt 480ctgcttgtgt ctgatattgg cacaattgtg tggggcgcca
cttccgccat ggccaccgga 540tacgtcaagg tcatcttctt ctgcctgggt
ctgtgttatg gtgctaacac gttctttcac 600gctgccaagg cctacatcga
gggttaccac accgtgccga agggccggtg tcgccaggtg 660gtgactggca
tggcttggct cttcttcgta tcatggggta tgttccccat cctgttcatc
720ctcggccccg agggcttcgg cgtcctgagc gtgtacggct ccaccgtcgg
ccacaccatc 780attgacctga tgtcgaagaa ctgctggggt ctgctcggcc
actacctgcg cgtgctgatc 840cacgagcata tcctcatcca cggcgacatt
cgcaagacca ccaaattgaa cattggtggc 900actgagattg aggtcgagac
gctggtggag gacgaggccg aggctggcgc ggtcaacaag 960ggcaccggca
agtacgcctc ccgcgagtcc ttcctggtca tgcgcgacaa gatgaaggag
1020aagggcattg acgtgcgcgc ctctctggac aacagcaagg aggtggagca
ggagcaggcc 1080gccagggctg ccatgatgat gatgaacggc aatggcatgg
gtatgggaat gggaatgaac 1140ggcatgaacg gaatgggcgg tatgaacggg
atggctggcg gcgccaagcc cggcctggag 1200ctcactccgc agctacagcc
cggccgcgtc atcctggcgg tgccggacat cagcatggtt 1260gacttcttcc
gcgagcagtt tgctcagcta tcggtgacgt acgagctggt gccggccctg
1320ggcgctgaca acacactggc gctggttacg caggcgcaga acctgggcgg
cgtggacttt 1380gtgttgattc accccgagtt cctgcgcgac cgctctagca
ccagcatcct gagccgcctg 1440cgcggcgcgg gccagcgtgt ggctgcgttc
ggctgggcgc agctggggcc catgcgtgac 1500ctgatcgagt ccgcaaacct
ggacggctgg ctggagggcc cctcgttcgg acagggcatc 1560ctgccggccc
acatcgttgc cctggtggcc aagatgcagc agatgcgcaa gatgcagcag
1620atgcagcaga ttggcatgat gaccggcggc atgaacggca tgggcggcgg
tatgggcggc 1680ggcatgaacg gcatgggcgg cggcaacggc atgaacaaca
tgggcaacgg catgggcggc 1740ggcatgggca acggcatggg cggcaatggc
atgaacggaa tgggtggcgg caacggcatg 1800aacaacatgg gcggcaacgg
aatggccggc aacggaatgg gcggcggcat gggcggcaac 1860ggtatgggtg
gctccatgaa cggcatgagc tccggcgtgg tggccaacgt gacgccctcc
1920gccgccggcg gcatgggcgg catgatgaac ggcggcatgg ctgcgcccca
gtcgcccggc 1980atgaacggcg gccgcctggg taccaacccg ctcttcaacg
ccgcgccctc accgctcagc 2040tcgcagctcg gtgccgaggc aggcatgggc
agcatgggag gcatgggcgg aatgagcgga 2100atgggaggca tgggtggaat
ggggggcatg ggcggcgccg gcgccgccac gacgcaggct 2160gcgggcggca
acgcggaggc ggagatgctg cagaatctca tgaacgagat caatcgcctg
2220aagcgcgagc ttggcgagta a 22414737PRTChlamydomonas reinhardtii
4Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5
10 15Val Thr Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro Glu
Asp 20 25 30Gln Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn
Gly Ala 35 40 45Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly
Phe Ser Ile 50 55 60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys
Ser Thr Cys Gly65 70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu
Met Val Lys Val Ile Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro
Ser Met Leu Tyr Leu Ala Thr 100 105 110Gly His Arg Val Gln Trp Leu
Arg Tyr Ala Glu Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Leu Ile
His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg
Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly Thr Ile145 150 155
160Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile
165 170 175Phe Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe
His Ala 180 185 190Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro
Lys Gly Arg Cys 195 200 205Arg Gln Val Val Thr Gly Met Ala Trp Leu
Phe Phe Val Ser Trp Gly 210 215 220Met Phe Pro Ile Leu Phe Ile Leu
Gly Pro Glu Gly Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser
Thr Val Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys
Trp Gly Leu Leu Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu
His Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280
285Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala
290 295 300Glu Ala Gly Ala Val Asn Lys Gly Thr Gly Lys Tyr Ala Ser
Arg Glu305 310 315 320Ser Phe Leu Val Met Arg Asp Lys Met Lys Glu
Lys Gly Ile Asp Val 325 330 335Arg Ala Ser Leu Asp Asn Ser Lys Glu
Val Glu Gln Glu Gln Ala Ala 340 345 350Arg Ala Ala Met Met Met Met
Asn Gly Asn Gly Met Gly Met Gly Met 355 360 365Gly Met Asn Gly Met
Asn Gly Met Gly Gly Met Asn Gly Met Ala Gly 370 375 380Gly Ala Lys
Pro Gly Leu Glu Leu Thr Pro Gln Leu Gln Pro Gly Arg385 390 395
400Val Ile Leu Ala Val Pro Asp Ile Ser Met Val Asp Phe Phe Arg Glu
405 410 415Gln Phe Ala Gln Leu Ser Val Thr Tyr Glu Leu Val Pro Ala
Leu Gly 420 425 430Ala Asp Asn Thr Leu Ala Leu Val Thr Gln Ala Gln
Asn Leu Gly Gly 435 440 445Val Asp Phe Val Leu Ile His Pro Glu Phe
Leu Arg Asp Arg Ser Ser 450 455 460Thr Ser Ile Leu Ser Arg Leu Arg
Gly Ala Gly Gln Arg Val Ala Ala465 470 475 480Phe Gly Trp Ala Gln
Leu Gly Pro Met Arg Asp Leu Ile Glu Ser Ala 485 490 495Asn Leu Asp
Gly Trp Leu Glu Gly Pro Ser Phe Gly Gln Gly Ile Leu 500 505 510Pro
Ala His Ile Val Ala Leu Val Ala Lys Met Gln Gln Met Arg Lys 515 520
525Met Gln Gln Met Gln Gln Ile Gly Met Met Thr Gly Gly Met Asn Gly
530 535 540Met Gly Gly Gly Met Gly Gly Gly Met Asn Gly Met Gly Gly
Gly Asn545 550 555 560Gly Met Asn Asn Met Gly Asn Gly Met Gly Gly
Gly Met Gly Asn Gly 565 570 575Met Gly Gly Asn Gly Met Asn Gly Met
Gly Gly Gly Asn Gly Met Asn 580 585 590Asn Met Gly Gly Asn Gly Met
Ala Gly Asn Gly Met Gly Gly Gly Met 595 600 605Gly Gly Asn Gly Met
Gly Gly Ser Met Asn Gly Met Ser Ser Gly Val 610 615 620Val Ala Asn
Val Thr Pro Ser Ala Ala Gly Gly Met Gly Gly Met Met625 630 635
640Asn Gly Gly Met Ala Ala Pro Gln Ser Pro Gly Met Asn Gly Gly Arg
645 650 655Leu Gly Thr Asn Pro Leu Phe Asn Ala Ala Pro Ser Pro Leu
Ser Ser 660 665 670Gln Leu Gly Ala Glu Ala Gly Met Gly Ser Met Gly
Gly Met Gly Gly 675 680 685Met Ser Gly Met Gly Gly Met Gly Gly Met
Gly Gly Met Gly Gly Ala 690 695 700Gly Ala Ala Thr Thr Gln Ala Ala
Gly Gly Asn Ala Glu Ala Glu Met705 710 715 720Leu Gln Asn Leu Met
Asn Glu Ile Asn Arg Leu Lys Arg Glu Leu Gly 725 730
735Glu53599DNAChlamydomonas reinhardtii 5ttgacatctg tcgccaagca
agcattaaac
atggattatg gaggcgccct gagtgccgtt 60gggcgcgagc tgctatttgt aacgaaccca
gtagtcgtca atggctctgt acttgtgcct 120gaggaccagt gttactgcgc
gggctggatt gagtcgcgtg gcacaaacgg tgcccaaacg 180gcgtcgaacg
tgctgcaatg gcttgctgct ggcttctcca tcctactgct tatgttttac
240gcctaccaaa catggaagtc aacctgcggc tgggaggaga tctatgtgtg
cgctatcgag 300atggtcaagg tgattctcga gttcttcttc gagtttaaga
acccgtccat gctgtatcta 360gccacaggcc accgcgtcca gtggttgcgt
tacgccgagt ggcttctcac ctgcccggtc 420attctcattc acctgtcaaa
cctgacgggc ttgtccaacg actacagcag gcgcaccatg 480ggtctgcttg
tgtctgatat tggcacaatt gtgtggggcg ccacttccgc catggccacc
540ggatacgtca aggtcatctt cttctgcctg ggtctgtgtt atggtgctaa
cacgttcttt 600cacgctgcca aggcctacat cgagggttac cacaccgtgc
cgaagggccg gtgtcgccag 660gtggtgactg gcatggcttg gctcttcttc
gtatcatggg gtatgttccc catcctgttc 720atcctcggcc ccgagggctt
cggcgtcctg agcgtgtacg gctccaccgt cggccacacc 780atcattgacc
tgatgtcgaa gaactgctgg ggtctgctcg gccactacct gcgcgtgctg
840atccacgagc atatcctcat ccacggcgac attcgcaaga ccaccaaatt
gaacattggt 900ggcactgaga ttgaggtcga gacgctggtg gaggacgagg
ccgaggctgg cgcggtcaac 960aagggcaccg gcaagtacgc ctcccgcgag
tccttcctgg tcatgcgcga caagatgaag 1020gagaagggca ttgacgtgcg
cgcctctctg gacaacagca aggaggtgga gcaggagcag 1080gccgccaggg
ctgccatgat gatgatgaac ggcaatggca tgggtatggg aatgggaatg
1140aacggcatga acggaatggg cggtatgaac gggatggctg gcggcgccaa
gcccggcctg 1200gagctcactc cgcagctaca gcccggccgc gtcatcctgg
cggtgccgga catcagcatg 1260gttgacttct tccgcgagca gtttgctcag
ctatcggtga cgtacgagct ggtgccggcc 1320ctgggcgctg acaacacact
ggcgctggtt acgcaggcgc agaacctggg cggcgtggac 1380tttgtgttga
ttcaccccga gttcctgcgc gaccgctcta gcaccagcat cctgagccgc
1440ctgcgcggcg cgggccagcg tgtggctgcg ttcggctggg cgcagctggg
gcccatgcgt 1500gacctgatcg agtccgcaaa cctggacggc tggctggagg
gcccctcgtt cggacagggc 1560atcctgccgg cccacatcgt tgccctggtg
gccaagatgc agcagatgcg caagatgcag 1620cagatgcagc agattggcat
gatgaccggc ggcatgaacg gcatgggcgg cggtatgggc 1680ggcggcatga
acggcatggg cggcggcaac ggcatgaaca acatgggcaa cggcatgggc
1740ggcggcatgg gcaacggcat gggcggcaat ggcatgaacg gaatgggtgg
cggcaacggc 1800atgaacaaca tgggcggcaa cggaatggcc ggcaacggaa
tgggcggcgg catgggcggc 1860aacggtatgg gtggctccat gaacggcatg
agctccggcg tggtggccaa cgtgacgccc 1920tccgccgccg gcggcatggg
cggcatgatg aacggcggca tggctgcgcc ccagtcgccc 1980ggcatgaacg
gcggccgcct gggtaccaac ccgctcttca acgccgcgcc ctcaccgctc
2040agctcgcagc tcggtgccga ggcaggcatg ggcagcatgg gaggcatggg
cggaatgagc 2100ggaatgggag gcatgggtgg aatggggggc atgggcggcg
ccggcgccgc cacgacgcag 2160gctgcgggcg gcaacgcgga ggcggagatg
ctgcagaatc tcatgaacga gatcaatcgc 2220ctgaagcgcg agcttggcga
gtaaaaggct ggaggccggt actgcgatac ctgcgagctc 2280gcgcgcctga
ctcgtcgtac acacggctca ggagcacgcg cgcgtggact tctcaacctg
2340tgtgcaacgt atctagagcg gcctgtgcgc gaccgtccgt gagcattccg
gtgcgatctt 2400cccgccttcg caccgcaagt tcccttcctg gccctgctgc
gcctgacgca tcgtccgaac 2460ggaagggcgg cttgatcagt aaagcattga
agactgaagt cgtgcgaccg tagtgctatg 2520gctctgcacg taagtgggcg
ctgccctgct tactacgcat tgcccaagac tgcttccttt 2580tggtggccga
ggccctggtc ccacatcatt catttgcata acgtactgtt tagttacata
2640cgctttgctt aacctcgaca attgcaacat gggctgagag tccgtacggc
ggctatggac 2700gaaggtgtta tcggatgtga ttaggaatct cggttgaaag
gcttcgagaa agtgagcttc 2760ttctgtggct tctgttgggg tcatcaagaa
gaacgacggt aaggcaaacg aggtaaaagt 2820ggcacgtctt tgtgcacaac
gggcccgtgg agagtggggg agtgcatgtg tgcggtccta 2880acacgcgagt
gcaaagcggg cttttctgga gctgggttac ggtctggctc ggcaactgct
2940ctgtgtttta accacagctt cggaagtctg ggtatgtttt gttggcagaa
acatttgggt 3000aacttgaggg tgattcgtct ggagtcggac aacatggctg
ccgtccgtgt gcagggacgg 3060taatcaatga agctgaagct gtgatgctca
ccacacgttg catacccctg cttacaaaaa 3120cactttgatg tcgtggccaa
actatgcgtg agcaaagagt taaagaggca tgagtgcatg 3180gttgcggacg
tgcgcaacaa ttgcatcaag tatttgacgc cttcaagcca acaagtgcgc
3240gcgcggcaac ttgattaaca cgccggacgc agtggtgggg gcgtgtacag
tgtttatgag 3300ctgccattct gcgatccgta gtgttaggtt gcgtgtgacg
ccgcgcggct gtgggccctt 3360acatggagag ttgggtgctt caccacacgg
ttggcgccgc tgaagggtgt gctatgtttt 3420ggtaaagccg gggccctgaa
gaccgcaacc gtagaaccgt actgaaaggg tgtcagcccg 3480gggtaactgg
atgccctggg acatagctat taatgttgaa gtgaagccgt caagccgagt
3540gccgtgcgcc gctgtatcac caaggcccgt ccaaaaaaaa aaaaaaaaaa
aaaaaaaaa 35996737PRTChlamydomonas reinhardtii 6Met Asp Tyr Gly Gly
Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn Pro
Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln Cys Tyr
Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln Thr
Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60Leu
Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75
80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile Leu
85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala
Thr 100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu
Leu Thr Cys 115 120 125Pro Val Ile Leu Ile His Leu Ser Asn Leu Thr
Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met Gly Leu Leu
Val Ser Asp Ile Gly Thr Ile145 150 155 160Val Trp Gly Ala Thr Ser
Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe Phe Cys Leu
Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190Ala Lys
Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200
205Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly
210 215 220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly
Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile
Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu Gly His
Tyr Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile His Gly
Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly Thr Glu
Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu Ala Gly
Ala Val Asn Lys Gly Thr Gly Lys Tyr Ala Ser Arg Glu305 310 315
320Ser Phe Leu Val Met Arg Asp Lys Met Lys Glu Lys Gly Ile Asp Val
325 330 335Arg Ala Ser Leu Asp Asn Ser Lys Glu Val Glu Gln Glu Gln
Ala Ala 340 345 350Arg Ala Ala Met Met Met Met Asn Gly Asn Gly Met
Gly Met Gly Met 355 360 365Gly Met Asn Gly Met Asn Gly Met Gly Gly
Met Asn Gly Met Ala Gly 370 375 380Gly Ala Lys Pro Gly Leu Glu Leu
Thr Pro Gln Leu Gln Pro Gly Arg385 390 395 400Val Ile Leu Ala Val
Pro Asp Ile Ser Met Val Asp Phe Phe Arg Glu 405 410 415Gln Phe Ala
Gln Leu Ser Val Thr Tyr Glu Leu Val Pro Ala Leu Gly 420 425 430Ala
Asp Asn Thr Leu Ala Leu Val Thr Gln Ala Gln Asn Leu Gly Gly 435 440
445Val Asp Phe Val Leu Ile His Pro Glu Phe Leu Arg Asp Arg Ser Ser
450 455 460Thr Ser Ile Leu Ser Arg Leu Arg Gly Ala Gly Gln Arg Val
Ala Ala465 470 475 480Phe Gly Trp Ala Gln Leu Gly Pro Met Arg Asp
Leu Ile Glu Ser Ala 485 490 495Asn Leu Asp Gly Trp Leu Glu Gly Pro
Ser Phe Gly Gln Gly Ile Leu 500 505 510Pro Ala His Ile Val Ala Leu
Val Ala Lys Met Gln Gln Met Arg Lys 515 520 525Met Gln Gln Met Gln
Gln Ile Gly Met Met Thr Gly Gly Met Asn Gly 530 535 540Met Gly Gly
Gly Met Gly Gly Gly Met Asn Gly Met Gly Gly Gly Asn545 550 555
560Gly Met Asn Asn Met Gly Asn Gly Met Gly Gly Gly Met Gly Asn Gly
565 570 575Met Gly Gly Asn Gly Met Asn Gly Met Gly Gly Gly Asn Gly
Met Asn 580 585 590Asn Met Gly Gly Asn Gly Met Ala Gly Asn Gly Met
Gly Gly Gly Met 595 600 605Gly Gly Asn Gly Met Gly Gly Ser Met Asn
Gly Met Ser Ser Gly Val 610 615 620Val Ala Asn Val Thr Pro Ser Ala
Ala Gly Gly Met Gly Gly Met Met625 630 635 640Asn Gly Gly Met Ala
Ala Pro Gln Ser Pro Gly Met Asn Gly Gly Arg 645 650 655Leu Gly Thr
Asn Pro Leu Phe Asn Ala Ala Pro Ser Pro Leu Ser Ser 660 665 670Gln
Leu Gly Ala Glu Ala Gly Met Gly Ser Met Gly Gly Met Gly Gly 675 680
685Met Ser Gly Met Gly Gly Met Gly Gly Met Gly Gly Met Gly Gly Ala
690 695 700Gly Ala Ala Thr Thr Gln Ala Ala Gly Gly Asn Ala Glu Ala
Glu Met705 710 715 720Leu Gln Asn Leu Met Asn Glu Ile Asn Arg Leu
Lys Arg Glu Leu Gly 725 730 735Glu72448DNAChlamydomonas reinhardtii
7catctgtcgc caagcaagca ttaaacatgg attatggagg cgccctgagt gccgttgggc
60gcgagctgct atttgtaacg aacccagtag tcgtcaatgg ctctgtactt gtgcctgagg
120accagtgtta ctgcgcgggc tggattgagt cgcgtggcac aaacggtgcc
caaacggcgt 180cgaacgtgct gcaatggctt gctgctggct tctccatcct
actgcttatg ttttacgcct 240accaaacatg gaagtcaacc tgcggctggg
aggagatcta tgtgtgcgct atcgagatgg 300tcaaggtgat tctcgagttc
ttcttcgagt ttaagaaccc gtccatgctg tatctagcca 360caggccaccg
cgtccagtgg ttgcgttacg ccgagtggct tctcacctgc ccggtcattc
420tcattcacct gtcaaacctg acgggcttgt ccaacgacta cagcaggcgc
accatgggtc 480tgcttgtgtc tgatattggc acaattgtgt ggggcgccac
ttccgccatg gccaccggat 540acgtcaaggt catcttcttc tgcctgggtc
tgtgttatgg tgctaacacg ttctttcacg 600ctgccaaggc ctacatcgag
ggttaccaca ccgtgccgaa gggccggtgt cgccaggtgg 660tgactggcat
ggcttggctc ttcttcgtat catggggtat gttccccatc ctgttcatcc
720tcggccccga gggcttcggc gtcctgagcg tgtacggctc caccgtcggc
cacaccatca 780ttgacctgat gtcgaagaac tgctggggtc tgctcggcca
ctacctgcgc gtgctgatcc 840acgagcatat cctcatccac ggcgacattc
gcaagaccac caaattgaac attggtggca 900ctgagattga ggtcgagacg
ctggtggagg acgaggccga ggctggcgcg gtcaacaagg 960gcaccggcaa
gtacgcctcc cgcgagtcct tcctggtcat gcgcgacaag atgaaggaga
1020agggcattga cgtgcgcgcc tctctggaca acagcaagga ggtggagcag
gagcaggccg 1080ccagggctgc catgatgatg atgaacggca atggcatggg
tatgggaatg ggaatgaacg 1140gcatgaacgg aatgggcggt atgaacggga
tggctggcgg cgccaagccc ggcctggagc 1200tcactccgca gctacagccc
ggccgcgtca tcctggcggt gccggacatc agcatggttg 1260acttcttccg
cgagcagttt gctcagctat cggtgacgta cgagctggtg ccggccctgg
1320gcgctgacaa cacactggcg ctggttacgc aggcgcagaa cctgggcggc
gtggactttg 1380tgttgattca ccccgagttc ctgcgcgacc gctctagcac
cagcatcctg agccgcctgc 1440gcggcgcggg ccagcgtgtg gctgcgttcg
gctgggcgca gctggggccc atgcgtgacc 1500tgatcgagtc cgcaaacctg
gacggctggc tggagggccc ctcgttcgga cagggcatcc 1560tgccggccca
catcgttgcc ctggtggcca agatgcagca gatgcgcaag atgcagcaga
1620tgcagcagat tggcatgatg accggcggca tgaacggcat gggcggcggt
atgggcggcg 1680gcatgaacgg catgggcggc ggcaacggca tgaacaacat
gggcaacggc atgggcggcg 1740gcatgggcaa cggcatgggc ggcaatggca
tgaacggaat gggtggcggc aacggcatga 1800acaacatggg cggcaacgga
atggccggca acggaatggg cggcggcatg ggcggcaacg 1860gtatgggtgg
ctccatgaac ggcatgagct ccggcgtggt ggccaacgtg acgccctccg
1920ccgccggcgg catgggcggc atgatgaacg gcggcatggc tgcgccccag
tcgcccggca 1980tgaacggcgg ccgcctgggt accaacccgc tcttcaacgc
cgcgccctca ccgctcagct 2040cgcagctcgg tgccgaggca ggcatgggca
gcatgggagg catgggcgga atgagcggaa 2100tgggaggcat gggtggaatg
gggggcatgg gcggcgccgg cgccgccacg acgcaggctg 2160cgggcggcaa
cgcggaggcg gagatgctgc agaatctcat gaacgagatc aatcgcctga
2220agcgcgagct tggcgagtaa aaggctggag gccggtactg cgatacctgc
gagctcgcgc 2280gcctgactcg tcgtacacac ggctcaggag cacgcgcgcg
tggacttctc aacctgtgtg 2340caacgtatct agagcggcct gtgcgcgacc
gtccgtgagc attccggtgc gatcttcccg 2400ccttcgcacc gcaagttccc
ttcctggccc tgctgcgcct gacgcatc 24488737PRTChlamydomonas reinhardtii
8Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5
10 15Val Thr Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro Glu
Asp 20 25 30Gln Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn
Gly Ala 35 40 45Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly
Phe Ser Ile 50 55 60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys
Ser Thr Cys Gly65 70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu
Met Val Lys Val Ile Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro
Ser Met Leu Tyr Leu Ala Thr 100 105 110Gly His Arg Val Gln Trp Leu
Arg Tyr Ala Glu Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Leu Ile
His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg
Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly Thr Ile145 150 155
160Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile
165 170 175Phe Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe
His Ala 180 185 190Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro
Lys Gly Arg Cys 195 200 205Arg Gln Val Val Thr Gly Met Ala Trp Leu
Phe Phe Val Ser Trp Gly 210 215 220Met Phe Pro Ile Leu Phe Ile Leu
Gly Pro Glu Gly Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser
Thr Val Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys
Trp Gly Leu Leu Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu
His Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280
285Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala
290 295 300Glu Ala Gly Ala Val Asn Lys Gly Thr Gly Lys Tyr Ala Ser
Arg Glu305 310 315 320Ser Phe Leu Val Met Arg Asp Lys Met Lys Glu
Lys Gly Ile Asp Val 325 330 335Arg Ala Ser Leu Asp Asn Ser Lys Glu
Val Glu Gln Glu Gln Ala Ala 340 345 350Arg Ala Ala Met Met Met Met
Asn Gly Asn Gly Met Gly Met Gly Met 355 360 365Gly Met Asn Gly Met
Asn Gly Met Gly Gly Met Asn Gly Met Ala Gly 370 375 380Gly Ala Lys
Pro Gly Leu Glu Leu Thr Pro Gln Leu Gln Pro Gly Arg385 390 395
400Val Ile Leu Ala Val Pro Asp Ile Ser Met Val Asp Phe Phe Arg Glu
405 410 415Gln Phe Ala Gln Leu Ser Val Thr Tyr Glu Leu Val Pro Ala
Leu Gly 420 425 430Ala Asp Asn Thr Leu Ala Leu Val Thr Gln Ala Gln
Asn Leu Gly Gly 435 440 445Val Asp Phe Val Leu Ile His Pro Glu Phe
Leu Arg Asp Arg Ser Ser 450 455 460Thr Ser Ile Leu Ser Arg Leu Arg
Gly Ala Gly Gln Arg Val Ala Ala465 470 475 480Phe Gly Trp Ala Gln
Leu Gly Pro Met Arg Asp Leu Ile Glu Ser Ala 485 490 495Asn Leu Asp
Gly Trp Leu Glu Gly Pro Ser Phe Gly Gln Gly Ile Leu 500 505 510Pro
Ala His Ile Val Ala Leu Val Ala Lys Met Gln Gln Met Arg Lys 515 520
525Met Gln Gln Met Gln Gln Ile Gly Met Met Thr Gly Gly Met Asn Gly
530 535 540Met Gly Gly Gly Met Gly Gly Gly Met Asn Gly Met Gly Gly
Gly Asn545 550 555 560Gly Met Asn Asn Met Gly Asn Gly Met Gly Gly
Gly Met Gly Asn Gly 565 570 575Met Gly Gly Asn Gly Met Asn Gly Met
Gly Gly Gly Asn Gly Met Asn 580 585 590Asn Met Gly Gly Asn Gly Met
Ala Gly Asn Gly Met Gly Gly Gly Met 595 600 605Gly Gly Asn Gly Met
Gly Gly Ser Met Asn Gly Met Ser Ser Gly Val 610 615 620Val Ala Asn
Val Thr Pro Ser Ala Ala Gly Gly Met Gly Gly Met Met625 630 635
640Asn Gly Gly Met Ala Ala Pro Gln Ser Pro Gly Met Asn Gly Gly Arg
645 650 655Leu Gly Thr Asn Pro Leu Phe Asn Ala Ala Pro Ser Pro Leu
Ser Ser 660 665 670Gln Leu Gly Ala Glu Ala Gly Met Gly Ser Met Gly
Gly Met Gly Gly 675 680 685Met Ser Gly Met Gly Gly Met Gly Gly Met
Gly Gly Met Gly Gly Ala 690 695 700Gly Ala Ala Thr Thr Gln Ala Ala
Gly Gly Asn Ala Glu Ala Glu Met705 710 715 720Leu Gln Asn Leu Met
Asn Glu Ile Asn Arg Leu Lys Arg Glu Leu Gly
725 730 735Glu93033DNAArtificial SequenceChR2 mutant 9atggattacc
ctgtggcccg gtccctgatt gtaagatacc ccaccgatct gggcaatgga 60accgtgtgca
tgcccagagg acaatgctac tgcgaggggt ggctgaggag ccggggcact
120agtatcgaaa aaaccatcgc tatcaccctc cagtgggtag tgttcgctct
gtccgtagcc 180tgtctcggct ggtatgcata ccaagcctgg agggctacct
gtgggtggga ggaagtatac 240gtggccctga tcgagatgat gaagtccatc
atcgaggctt tccatgagtt cgactcccca 300gccacactct ggctcagcag
tgggaatggc gtagtgtgga tgagatatgg agagtggctg 360ctgacctgtc
ccgtcctgct cattcatctg tccaatctga ccgggctgaa agatgactac
420tccaagagaa caatgggact gctggtgagt gacgtggggt gtattgtgtg
gggagccacc 480tccgccatgt gcactggatg gaccaagatc ctctttttcc
tgatttccct ctcctatggg 540atgtatacat acttccacgc cgctaaggtg
tatattgagg ccttccacac tgtacctaaa 600ggcatctgta gggagctcgt
gcgggtgatg gcatggacct tctttgtggc ctgggggatg 660ttccccgtgc
tgttcctcct cggcactgag ggatttggcc acattagtcc ttacgggtcc
720gcaattggac actccatcct ggatctgatt gccaagaata tgtggggggt
gctgggaaat 780tatctgcggg taaagatcca cgagcatatc ctgctgtatg
gcgatatcag aaagaagcag 840aaaatcacca ttgctggaca ggaaatggag
gtggagacac tggtagcaga ggaggaggac 900gggaccgcgg tcgccaccat
ggtgtctaag ggcgaagagc tgattaagga gaacatgcac 960atgaagctgt
acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc
1020gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
gggcggccct 1080ctccccttcg ccttcgacat cctggctacc agcttcatgt
acggcagcaa aaccttcatc 1140aaccacaccc agggcatccc cgacttcttt
aagcagtcct tccctgaggg cttcacatgg 1200gagagagtca ccacatacga
agacgggggc gtgctgaccg ctacccagga caccagcctc 1260caggacggct
gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc
1320cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagatgct
gtaccccgct 1380gacggcggcc tggaaggcag agccgacatg gccctgaagc
tcgtgggcgg gggccacctg 1440atctgcaact tgaagaccac atacagatcc
aagaaacccg ctaagaacct caagatgccc 1500ggcgtctact atgtggacag
aagactggaa agaatcaagg aggccgacaa agagacctac 1560gtcgagcagc
acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac
1620aaacttaatt gcctgcagga gaagaagtca tgcagccagc gcatggccga
attccggcaa 1680tactgttgga acccggacac tgggcagatg ctgggccgca
ccccagcccg gtgggtgtgg 1740atcagcctgt actatgcagc tttctacgtg
gtcatgactg ggctctttgc cttgtgcatc 1800tatgtgctga tgcagaccat
tgatccctac acccccgact accaggacca gttaaagtca 1860ccgggggtaa
ccttgagacc ggatgtgtat ggggaaagag ggctgcagat ttcctacaac
1920atctctgaaa acagctctag acaggcccag atcaccggac gtccggagac
tgagacattg 1980ccaccggtgg actacggggg ggccctgagc gctgtgggca
gagaactcct gttcgtgaca 2040aatccagtcg tggtgaacgg ctccgtactc
gtacccgagg atcagtgcta ttgcgcagga 2100tggatcgaga gcagaggcac
aaacggcgca cagactgcat ccaacgtgct ccagtggttg 2160gccgcaggct
tttccattct cctgctcatg ttttacgcct accagacttg gaagtccaca
2220tgtggctggg aggaaatcta cgtgtgtgca atcgaaatgg tgaaggtgat
cctggagttt 2280ttcttcgaat ttaaaaaccc aagcatgctg tacctggcta
ctggccacag agtgcagtgg 2340ctgcggtatg ccgaatggct gctgacttgc
ccagtgattt gcatccacct gtccaacctg 2400actgggctgt ctaacgatta
cagtaggaga acaatgggac tgctcgtatc cgacatcggc 2460actatcgtat
ggggcgcaac tagtgccatg gccactggat acgtgaaagt gatcttcttc
2520tgcctgggac tctgctacgg agcaaacaca ttttttcatg ccgcaaaagc
atatatcgag 2580gggtatcata ccgtcccaaa gggccggtgt agacaagtgg
tgactggcat ggcttggctg 2640ttcttcgtgt cctgggggat gtttcccatc
ctctttatcc tgggcccaga aggcttcggg 2700gtgctgagtg tgtatggcag
taccgtagga cacactatca ttgacctgat gagcaaaaac 2760tgctgggggc
tgctcggcca ctacctgaga gtactcatcc acgagcatat cctgattcat
2820ggcgatatcc ggaaaactac caagctcaat atcgggggca ccgagattga
agtggagaca 2880ctcgtggagg acgaggccga ggccggagca gtgaacaaag
gcactggcaa gtatgcctcc 2940agagaatcct ttctggtgat gcgggacaaa
atgaaggaga aaggcattga tgtacggtgc 3000agtaatgcca aagccgtcga
gactgatgtg tag 3033101010PRTArtificial SequenceChR2 mutant 10Met
Asp Tyr Pro Val Ala Arg Ser Leu Ile Val Arg Tyr Pro Thr Asp1 5 10
15Leu Gly Asn Gly Thr Val Cys Met Pro Arg Gly Gln Cys Tyr Cys Glu
20 25 30Gly Trp Leu Arg Ser Arg Gly Thr Ser Ile Glu Lys Thr Ile Ala
Ile 35 40 45Thr Leu Gln Trp Val Val Phe Ala Leu Ser Val Ala Cys Leu
Gly Trp 50 55 60Tyr Ala Tyr Gln Ala Trp Arg Ala Thr Cys Gly Trp Glu
Glu Val Tyr65 70 75 80Val Ala Leu Ile Glu Met Met Lys Ser Ile Ile
Glu Ala Phe His Glu 85 90 95Phe Asp Ser Pro Ala Thr Leu Trp Leu Ser
Ser Gly Asn Gly Val Val 100 105 110Trp Met Arg Tyr Gly Glu Trp Leu
Leu Thr Cys Pro Val Leu Leu Ile 115 120 125His Leu Ser Asn Leu Thr
Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr 130 135 140Met Gly Leu Leu
Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr145 150 155 160Ser
Ala Met Cys Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser 165 170
175Leu Ser Tyr Gly Met Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile
180 185 190Glu Ala Phe His Thr Val Pro Lys Gly Ile Cys Arg Glu Leu
Val Arg 195 200 205Val Met Ala Trp Thr Phe Phe Val Ala Trp Gly Met
Phe Pro Val Leu 210 215 220Phe Leu Leu Gly Thr Glu Gly Phe Gly His
Ile Ser Pro Tyr Gly Ser225 230 235 240Ala Ile Gly His Ser Ile Leu
Asp Leu Ile Ala Lys Asn Met Trp Gly 245 250 255Val Leu Gly Asn Tyr
Leu Arg Val Lys Ile His Glu His Ile Leu Leu 260 265 270Tyr Gly Asp
Ile Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu 275 280 285Met
Glu Val Glu Thr Leu Val Ala Glu Glu Glu Asp Gly Thr Ala Val 290 295
300Ala Thr Met Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn Met
His305 310 315 320Met Lys Leu Tyr Met Glu Gly Thr Val Asn Asn His
His Phe Lys Cys 325 330 335Thr Ser Glu Gly Glu Gly Lys Pro Tyr Glu
Gly Thr Gln Thr Met Arg 340 345 350Ile Lys Val Val Glu Gly Gly Pro
Leu Pro Phe Ala Phe Asp Ile Leu 355 360 365Ala Thr Ser Phe Met Tyr
Gly Ser Lys Thr Phe Ile Asn His Thr Gln 370 375 380Gly Ile Pro Asp
Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp385 390 395 400Glu
Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln 405 410
415Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg
420 425 430Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys
Thr Leu 435 440 445Gly Trp Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala
Asp Gly Gly Leu 450 455 460Glu Gly Arg Ala Asp Met Ala Leu Lys Leu
Val Gly Gly Gly His Leu465 470 475 480Ile Cys Asn Leu Lys Thr Thr
Tyr Arg Ser Lys Lys Pro Ala Lys Asn 485 490 495Leu Lys Met Pro Gly
Val Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile 500 505 510Lys Glu Ala
Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala Val 515 520 525Ala
Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Lys Leu Asn Cys 530 535
540Leu Gln Glu Lys Lys Ser Cys Ser Gln Arg Met Ala Glu Phe Arg
Gln545 550 555 560Tyr Cys Trp Asn Pro Asp Thr Gly Gln Met Leu Gly
Arg Thr Pro Ala 565 570 575Arg Trp Val Trp Ile Ser Leu Tyr Tyr Ala
Ala Phe Tyr Val Val Met 580 585 590Thr Gly Leu Phe Ala Leu Cys Ile
Tyr Val Leu Met Gln Thr Ile Asp 595 600 605Pro Tyr Thr Pro Asp Tyr
Gln Asp Gln Leu Lys Ser Pro Gly Val Thr 610 615 620Leu Arg Pro Asp
Val Tyr Gly Glu Arg Gly Leu Gln Ile Ser Tyr Asn625 630 635 640Ile
Ser Glu Asn Ser Ser Arg Gln Ala Gln Ile Thr Gly Arg Pro Glu 645 650
655Thr Glu Thr Leu Pro Pro Val Asp Tyr Gly Gly Ala Leu Ser Ala Val
660 665 670Gly Arg Glu Leu Leu Phe Val Thr Asn Pro Val Val Val Asn
Gly Ser 675 680 685Val Leu Val Pro Glu Asp Gln Cys Tyr Cys Ala Gly
Trp Ile Glu Ser 690 695 700Arg Gly Thr Asn Gly Ala Gln Thr Ala Ser
Asn Val Leu Gln Trp Leu705 710 715 720Ala Ala Gly Phe Ser Ile Leu
Leu Leu Met Phe Tyr Ala Tyr Gln Thr 725 730 735Trp Lys Ser Thr Cys
Gly Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu 740 745 750Met Val Lys
Val Ile Leu Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser 755 760 765Met
Leu Tyr Leu Ala Thr Gly His Arg Val Gln Trp Leu Arg Tyr Ala 770 775
780Glu Trp Leu Leu Thr Cys Pro Val Ile Cys Ile His Leu Ser Asn
Leu785 790 795 800Thr Gly Leu Ser Asn Asp Tyr Ser Arg Arg Thr Met
Gly Leu Leu Val 805 810 815Ser Asp Ile Gly Thr Ile Val Trp Gly Ala
Thr Ser Ala Met Ala Thr 820 825 830Gly Tyr Val Lys Val Ile Phe Phe
Cys Leu Gly Leu Cys Tyr Gly Ala 835 840 845Asn Thr Phe Phe His Ala
Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr 850 855 860Val Pro Lys Gly
Arg Cys Arg Gln Val Val Thr Gly Met Ala Trp Leu865 870 875 880Phe
Phe Val Ser Trp Gly Met Phe Pro Ile Leu Phe Ile Leu Gly Pro 885 890
895Glu Gly Phe Gly Val Leu Ser Val Tyr Gly Ser Thr Val Gly His Thr
900 905 910Ile Ile Asp Leu Met Ser Lys Asn Cys Trp Gly Leu Leu Gly
His Tyr 915 920 925Leu Arg Val Leu Ile His Glu His Ile Leu Ile His
Gly Asp Ile Arg 930 935 940Lys Thr Thr Lys Leu Asn Ile Gly Gly Thr
Glu Ile Glu Val Glu Thr945 950 955 960Leu Val Glu Asp Glu Ala Glu
Ala Gly Ala Val Asn Lys Gly Thr Gly 965 970 975Lys Tyr Ala Ser Arg
Glu Ser Phe Leu Val Met Arg Asp Lys Met Lys 980 985 990Glu Lys Gly
Ile Asp Val Arg Cys Ser Asn Ala Lys Ala Val Glu Thr 995 1000
1005Asp Val 1010113033DNAArtificial SequenceChR2 mutant
11atggattacc ctgtggcccg gtccctgatt gtaagatacc ccaccgatct gggcaatgga
60accgtgtgca tgcccagagg acaatgctac tgcgaggggt ggctgaggag ccggggcact
120agtatcgaaa aaaccatcgc tatcaccctc cagtgggtag tgttcgctct
gtccgtagcc 180tgtctcggct ggtatgcata ccaagcctgg agggctacct
gtgggtggga ggaagtatac 240gtggccctga tcgagatgat gaagtccatc
atcgaggctt tccatgagtt cgactcccca 300gccacactct ggctcagcag
tgggaatggc gtagtgtgga tgagatatgg agagtggctg 360ctgacctgtc
ccgtcctgct cattcatctg tccaatctga ccgggctgaa agatgactac
420tccaagagaa caatgggact gctggtgagt gacgtggggt gtattgtgtg
gggagccacc 480tccgccatgt gcactggatg gaccaagatc ctctttttcc
tgatttccct ctcctatggg 540atgtatacat acttccacgc cgctaaggtg
tatattgagg ccttccacac tgtacctaaa 600ggcatctgta gggagctcgt
gcgggtgatg gcatggacct tctttgtggc ctgggggatg 660ttccccgtgc
tgttcctcct cggcactgag ggatttggcc acattagtcc ttacgggtcc
720gcaattggac actccatcct ggatctgatt gccaagaata tgtggggggt
gctgggaaat 780tatctgcggg taaagatcca cgagcatatc ctgctgtatg
gcgatatcag aaagaagcag 840aaaatcacca ttgctggaca ggaaatggag
gtggagacac tggtagcaga ggaggaggac 900gggaccgcgg tcgccaccat
ggtgtctaag ggcgaagagc tgattaagga gaacatgcac 960atgaagctgt
acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc
1020gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga
gggcggccct 1080ctccccttcg ccttcgacat cctggctacc agcttcatgt
acggcagcaa aaccttcatc 1140aaccacaccc agggcatccc cgacttcttt
aagcagtcct tccctgaggg cttcacatgg 1200gagagagtca ccacatacga
agacgggggc gtgctgaccg ctacccagga caccagcctc 1260caggacggct
gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc
1320cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagatgct
gtaccccgct 1380gacggcggcc tggaaggcag agccgacatg gccctgaagc
tcgtgggcgg gggccacctg 1440atctgcaact tgaagaccac atacagatcc
aagaaacccg ctaagaacct caagatgccc 1500ggcgtctact atgtggacag
aagactggaa agaatcaagg aggccgacaa agagacctac 1560gtcgagcagc
acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac
1620aaacttaatt gcctgcagga gaagaagtca tgcagccagc gcatggccga
attccggcaa 1680tactgttgga acccggacac tgggcagatg ctgggccgca
ccccagcccg gtgggtgtgg 1740atcagcctgt actatgcagc tttctacgtg
gtcatgactg ggctctttgc cttgtgcatc 1800tatgtgctga tgcagaccat
tgatccctac acccccgact accaggacca gttaaagtca 1860ccgggggtaa
ccttgagacc ggatgtgtat ggggaaagag ggctgcagat ttcctacaac
1920atctctgaaa acagctctag acaggcccag atcaccggac gtccggagac
tgagacattg 1980ccaccggtgg actacggggg ggccctgagc gctgtgggca
gagaactcct gttcgtgaca 2040aatccagtcg tggtgaacgg ctccgtactc
gtacccgagg atcagtgcta ttgcgcagga 2100tggatcgaga gcagaggcac
aaacggcgca cagactgcat ccaacgtgct ccagtggttg 2160gccgcaggct
tttccattct cctgctcatg ttttacgcct accagacttg gaagtccaca
2220tgtggctggg aggaaatcta cgtgtgtgca atcgaaatgg tgaaggtgat
cctggagttt 2280ttcttcgaat ttaaaaaccc aagcatgctg tacctggcta
ctggccacag agtgcagtgg 2340ctgcggtatg ccgaatggct gctgacttgc
ccagtgattc tgatccacct gtccaacctg 2400actgggctgt ctaacgatta
cagtaggaga acaatgggac tgctcgtatc cgacatcggc 2460actatcgtat
ggggcgcaac tagtgccatg gccactggat acgtgaaagt gatcttcttc
2520tgcctgggac tctgctacgg agcaaacaca ttttttcatg ccgcaaaagc
atatatcgag 2580gggtatcata ccgtcccaaa gggccggtgt agacaagtgg
tgactggcat ggcttggctg 2640ttcttcgtgt cctgggggat gtttcccatc
ctctttatcc tgggcccaga aggcttcggg 2700gtgctgagtg tgtatggcag
taccgtagga cacactatca ttgacctgat gagcaaaaac 2760tgctgggggc
tgctcggcca ctacctgaga gtactcatcc acgagcatat cctgattcat
2820ggcgatatcc ggaaaactac caagctcaat atcgggggca ccgagattga
agtggagaca 2880ctcgtggagg acgaggccga ggccggagca gtgaacaaag
gcactggcaa gtatgcctcc 2940agagaatcct ttctggtgat gcgggacaaa
atgaaggaga aaggcattga tgtacggtgc 3000agtaatgcca aagccgtcga
gactgatgtg tag 3033121010PRTArtificial SequenceChR2 mutant
construct 12Met Asp Tyr Pro Val Ala Arg Ser Leu Ile Val Arg Tyr Pro
Thr Asp1 5 10 15Leu Gly Asn Gly Thr Val Cys Met Pro Arg Gly Gln Cys
Tyr Cys Glu 20 25 30Gly Trp Leu Arg Ser Arg Gly Thr Ser Ile Glu Lys
Thr Ile Ala Ile 35 40 45Thr Leu Gln Trp Val Val Phe Ala Leu Ser Val
Ala Cys Leu Gly Trp 50 55 60Tyr Ala Tyr Gln Ala Trp Arg Ala Thr Cys
Gly Trp Glu Glu Val Tyr65 70 75 80Val Ala Leu Ile Glu Met Met Lys
Ser Ile Ile Glu Ala Phe His Glu 85 90 95Phe Asp Ser Pro Ala Thr Leu
Trp Leu Ser Ser Gly Asn Gly Val Val 100 105 110Trp Met Arg Tyr Gly
Glu Trp Leu Leu Thr Cys Pro Val Leu Leu Ile 115 120 125His Leu Ser
Asn Leu Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr 130 135 140Met
Gly Leu Leu Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr145 150
155 160Ser Ala Met Cys Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile
Ser 165 170 175Leu Ser Tyr Gly Met Tyr Thr Tyr Phe His Ala Ala Lys
Val Tyr Ile 180 185 190Glu Ala Phe His Thr Val Pro Lys Gly Ile Cys
Arg Glu Leu Val Arg 195 200 205Val Met Ala Trp Thr Phe Phe Val Ala
Trp Gly Met Phe Pro Val Leu 210 215 220Phe Leu Leu Gly Thr Glu Gly
Phe Gly His Ile Ser Pro Tyr Gly Ser225 230 235 240Ala Ile Gly His
Ser Ile Leu Asp Leu Ile Ala Lys Asn Met Trp Gly 245 250 255Val Leu
Gly Asn Tyr Leu Arg Val Lys Ile His Glu His Ile Leu Leu 260 265
270Tyr Gly Asp Ile Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu
275 280 285Met Glu Val Glu Thr Leu Val Ala Glu Glu Glu Asp Gly Thr
Ala Val 290 295 300Ala Thr Met Val Ser Lys Gly Glu Glu Leu Ile Lys
Glu Asn Met His305 310 315 320Met Lys Leu Tyr Met Glu Gly Thr Val
Asn Asn His His Phe Lys Cys 325 330 335Thr Ser Glu Gly Glu Gly Lys
Pro Tyr Glu Gly Thr Gln Thr Met Arg 340 345 350Ile Lys Val Val Glu
Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu 355 360 365Ala Thr Ser
Phe Met Tyr Gly Ser Lys Thr Phe Ile Asn His Thr Gln 370 375 380Gly
Ile Pro Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp385 390
395 400Glu Arg Val Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr
Gln 405 410 415Asp Thr Ser Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val
Lys Ile Arg
420 425 430Gly Val Asn Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys
Thr Leu 435 440 445Gly Trp Glu Ala Ser Thr Glu Met Leu Tyr Pro Ala
Asp Gly Gly Leu 450 455 460Glu Gly Arg Ala Asp Met Ala Leu Lys Leu
Val Gly Gly Gly His Leu465 470 475 480Ile Cys Asn Leu Lys Thr Thr
Tyr Arg Ser Lys Lys Pro Ala Lys Asn 485 490 495Leu Lys Met Pro Gly
Val Tyr Tyr Val Asp Arg Arg Leu Glu Arg Ile 500 505 510Lys Glu Ala
Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala Val 515 520 525Ala
Arg Tyr Cys Asp Leu Pro Ser Lys Leu Gly His Lys Leu Asn Cys 530 535
540Leu Gln Glu Lys Lys Ser Cys Ser Gln Arg Met Ala Glu Phe Arg
Gln545 550 555 560Tyr Cys Trp Asn Pro Asp Thr Gly Gln Met Leu Gly
Arg Thr Pro Ala 565 570 575Arg Trp Val Trp Ile Ser Leu Tyr Tyr Ala
Ala Phe Tyr Val Val Met 580 585 590Thr Gly Leu Phe Ala Leu Cys Ile
Tyr Val Leu Met Gln Thr Ile Asp 595 600 605Pro Tyr Thr Pro Asp Tyr
Gln Asp Gln Leu Lys Ser Pro Gly Val Thr 610 615 620Leu Arg Pro Asp
Val Tyr Gly Glu Arg Gly Leu Gln Ile Ser Tyr Asn625 630 635 640Ile
Ser Glu Asn Ser Ser Arg Gln Ala Gln Ile Thr Gly Arg Pro Glu 645 650
655Thr Glu Thr Leu Pro Pro Val Asp Tyr Gly Gly Ala Leu Ser Ala Val
660 665 670Gly Arg Glu Leu Leu Phe Val Thr Asn Pro Val Val Val Asn
Gly Ser 675 680 685Val Leu Val Pro Glu Asp Gln Cys Tyr Cys Ala Gly
Trp Ile Glu Ser 690 695 700Arg Gly Thr Asn Gly Ala Gln Thr Ala Ser
Asn Val Leu Gln Trp Leu705 710 715 720Ala Ala Gly Phe Ser Ile Leu
Leu Leu Met Phe Tyr Ala Tyr Gln Thr 725 730 735Trp Lys Ser Thr Cys
Gly Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu 740 745 750Met Val Lys
Val Ile Leu Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser 755 760 765Met
Leu Tyr Leu Ala Thr Gly His Arg Val Gln Trp Leu Arg Tyr Ala 770 775
780Glu Trp Leu Leu Thr Cys Pro Val Ile Leu Ile His Leu Ser Asn
Leu785 790 795 800Thr Gly Leu Ser Asn Asp Tyr Ser Arg Arg Thr Met
Gly Leu Leu Val 805 810 815Ser Asp Ile Gly Thr Ile Val Trp Gly Ala
Thr Ser Ala Met Ala Thr 820 825 830Gly Tyr Val Lys Val Ile Phe Phe
Cys Leu Gly Leu Cys Tyr Gly Ala 835 840 845Asn Thr Phe Phe His Ala
Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr 850 855 860Val Pro Lys Gly
Arg Cys Arg Gln Val Val Thr Gly Met Ala Trp Leu865 870 875 880Phe
Phe Val Ser Trp Gly Met Phe Pro Ile Leu Phe Ile Leu Gly Pro 885 890
895Glu Gly Phe Gly Val Leu Ser Val Tyr Gly Ser Thr Val Gly His Thr
900 905 910Ile Ile Asp Leu Met Ser Lys Asn Cys Trp Gly Leu Leu Gly
His Tyr 915 920 925Leu Arg Val Leu Ile His Glu His Ile Leu Ile His
Gly Asp Ile Arg 930 935 940Lys Thr Thr Lys Leu Asn Ile Gly Gly Thr
Glu Ile Glu Val Glu Thr945 950 955 960Leu Val Glu Asp Glu Ala Glu
Ala Gly Ala Val Asn Lys Gly Thr Gly 965 970 975Lys Tyr Ala Ser Arg
Glu Ser Phe Leu Val Met Arg Asp Lys Met Lys 980 985 990Glu Lys Gly
Ile Asp Val Arg Cys Ser Asn Ala Lys Ala Val Glu Thr 995 1000
1005Asp Val 101013315PRTArtificial SequenceChR2 mutant construct
13Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1
5 10 15Val Thr Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro Glu
Asp 20 25 30Gln Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn
Gly Ala 35 40 45Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly
Phe Ser Ile 50 55 60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys
Ser Thr Cys Gly65 70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu
Met Val Lys Val Ile Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro
Ser Met Leu Tyr Leu Ala Thr 100 105 110Gly His Arg Val Gln Trp Leu
Arg Tyr Ala Glu Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Cys Ile
His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg
Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly Thr Ile145 150 155
160Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile
165 170 175Phe Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe
His Ala 180 185 190Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro
Lys Gly Arg Cys 195 200 205Arg Gln Val Val Thr Gly Met Ala Trp Leu
Phe Phe Val Ser Trp Gly 210 215 220Met Phe Pro Ile Leu Phe Ile Leu
Gly Pro Glu Gly Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser
Thr Val Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys
Trp Gly Leu Leu Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu
His Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280
285Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala
290 295 300Glu Ala Gly Ala Val Asn Lys Gly Thr Gly Lys305 310
31514315PRTArtificial SequenceChR2 mutant construct 14Met Asp Tyr
Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr
Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln
Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40
45Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile
50 55 60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys
Gly65 70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys
Val Ile Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu
Tyr Leu Ala Thr 100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala
Glu Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Leu Ile His Leu Ser
Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met
Gly Leu Leu Val Ser Asp Ile Gly Cys Ile145 150 155 160Val Trp Gly
Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe
Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185
190Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys
195 200 205Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser
Trp Gly 210 215 220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly
Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His
Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu
Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile
His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly
Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu
Ala Gly Ala Val Asn Lys Gly Thr Gly Lys305 310
31515945DNAArtificial SequenceChR2 mutant construct 15atggactacg
ggggggctct gtctgctgtc gggagggaac tgctgtttgt gactaaccct 60gtcgtcgtga
acgggagtgt gctggtccct gaggaccagt gctactgtgc cggctggatc
120gaatcacgcg gaaccaacgg ggcccagaca gctagcaatg tgctgcagtg
gctggccgct 180gggtttagta tcctgctgct gatgttctac gcctatcaga
cttggaagtc aacctgcggc 240tgggaggaaa tctacgtgtg cgctattgag
atggtgaaag tgatcctgga gttcttcttc 300gagttcaaga acccaagcat
gctgtacctg gctactggac accgagtgca gtggctgaga 360tatgcagaat
ggctgctgac atgccccgtc atctgcattc acctgtccaa cctgacaggc
420ctgagcaatg actactccag gagaactatg ggactgctgg tgtccgacat
cggctgcatt 480gtctggggag caacttctgc tatggcaacc ggatacgtga
aggtcatctt tttctgcctg 540gggctgtgct atggcgcaaa tacctttttc
cacgcagcca aggcctacat tgaggggtat 600cataccgtgc caaaaggccg
gtgccgacag gtggtcacag gaatggcttg gctgtttttc 660gtctcttggg
gaatgtttcc catcctgttc attctggggc ctgaagggtt cggcgtgctg
720tctgtctacg gaagtacagt ggggcatact atcattgacc tgatgtccaa
aaactgttgg 780ggcctgctgg gacactatct gagagtgctg atccacgagc
atatcctgat tcatggcgat 840attcggaaga ccacaaaact gaatatcggc
ggaaccgaga ttgaagtgga aacactggtg 900gaagacgagg ctgaggctgg
ggctgtgaac aaggggactg gcaaa 94516315PRTArtificial Sequencemutated
Chop2 16Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu
Phe1 5 10 15Val Thr Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro
Glu Asp 20 25 30Gln Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr
Asn Gly Ala 35 40 45Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala
Gly Phe Ser Ile 50 55 60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp
Lys Ser Thr Cys Gly65 70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile
Glu Met Val Lys Val Ile Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn
Pro Ser Met Leu Tyr Leu Ala Thr 100 105 110Gly His Arg Val Gln Trp
Leu Arg Tyr Ala Glu Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Cys
Ile His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser
Arg Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly Cys Ile145 150 155
160Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile
165 170 175Phe Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe
His Ala 180 185 190Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro
Lys Gly Arg Cys 195 200 205Arg Gln Val Val Thr Gly Met Ala Trp Leu
Phe Phe Val Ser Trp Gly 210 215 220Met Phe Pro Ile Leu Phe Ile Leu
Gly Pro Glu Gly Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser
Thr Val Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys
Trp Gly Leu Leu Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu
His Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280
285Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala
290 295 300Glu Ala Gly Ala Val Asn Lys Gly Thr Gly Lys305 310
31517315PRTArtificial Sequencemutated Chop2 17Met Asp Tyr Gly Gly
Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn Pro
Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln Cys Tyr
Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln Thr
Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60Leu
Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75
80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile Leu
85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala
Thr 100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu
Leu Thr Cys 115 120 125Pro Val Ile Leu Ile His Leu Ser Asn Leu Thr
Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met Gly Leu Leu
Val Ser Asp Ile Gly Ser Ile145 150 155 160Val Trp Gly Ala Thr Ser
Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe Phe Cys Leu
Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190Ala Lys
Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200
205Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly
210 215 220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly
Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile
Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu Gly His
Tyr Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile His Gly
Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly Thr Glu
Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu Ala Gly
Ala Val Asn Lys Gly Thr Gly Lys305 310 31518945DNAArtificial
Sequencemutated Chop2 18atggactacg ggggggctct gtctgctgtc gggagggaac
tgctgtttgt gactaaccct 60gtcgtcgtga acgggagtgt gctggtccct gaggaccagt
gctactgtgc cggctggatc 120gaatcacgcg gaaccaacgg ggcccagaca
gctagcaatg tgctgcagtg gctggccgct 180gggtttagta tcctgctgct
gatgttctac gcctatcaga cttggaagtc aacctgcggc 240tgggaggaaa
tctacgtgtg cgctattgag atggtgaaag tgatcctgga gttcttcttc
300gagttcaaga acccaagcat gctgtacctg gctactggac accgagtgca
gtggctgaga 360tatgcagaat ggctgctgac atgccccgtc atctgcattc
acctgtccaa cctgacaggc 420ctgagcaatg actactccag gagaactatg
ggactgctgg tgtccgacat cggcagcatt 480gtctggggag caacttctgc
tatggcaacc ggatacgtga aggtcatctt tttctgcctg 540gggctgtgct
atggcgcaaa tacctttttc cacgcagcca aggcctacat tgaggggtat
600cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg
gctgtttttc 660gtctcttggg gaatgtttcc catcctgttc attctggggc
ctgaagggtt cggcgtgctg 720tctgtctacg gaagtacagt ggggcatact
atcattgacc tgatgtccaa aaactgttgg 780ggcctgctgg gacactatct
gagagtgctg atccacgagc atatcctgat tcatggcgat 840attcggaaga
ccacaaaact gaatatcggc ggaaccgaga ttgaagtgga aacactggtg
900gaagacgagg ctgaggctgg ggctgtgaac aaggggactg gcaaa
94519315PRTArtificial Sequencemutated Chop2 19Met Asp Tyr Gly Gly
Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn Pro
Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln Cys Tyr
Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln Thr
Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60Leu
Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75
80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile Leu
85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala
Thr 100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu
Leu Thr Cys 115 120 125Pro Val Ile Cys Ile His Leu Ser Asn Leu Thr
Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met Gly Leu Leu
Val Ser Asp Ile Gly Ser Ile145 150 155 160Val Trp Gly Ala Thr Ser
Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe Phe Cys Leu
Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190Ala Lys
Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200
205Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly
210 215 220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly
Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile
Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu Gly His
Tyr Leu Arg Val Leu Ile His 260 265
270Glu His Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn
275 280 285Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp
Glu Ala 290 295 300Glu Ala Gly Ala Val Asn Lys Gly Thr Gly Lys305
310 31520315PRTArtificial Sequencemutated Chop2 20Met Asp Tyr Gly
Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn
Pro Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln Cys
Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln
Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55
60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65
70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile
Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu
Ala Thr 100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp
Leu Leu Thr Cys 115 120 125Pro Val Ile Ala Ile His Leu Ser Asn Leu
Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met Gly Leu
Leu Val Ser Asp Ile Gly Thr Ile145 150 155 160Val Trp Gly Ala Thr
Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe Phe Cys
Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190Ala
Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200
205Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly
210 215 220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly
Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile
Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu Gly His
Tyr Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile His Gly
Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly Thr Glu
Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu Ala Gly
Ala Val Asn Lys Gly Thr Gly Lys305 310 31521945DNAArtificial
Sequencemutated Chop2 21atggactacg ggggggctct gtctgctgtc gggagggaac
tgctgtttgt gactaaccct 60gtcgtcgtga acgggagtgt gctggtccct gaggaccagt
gctactgtgc cggctggatc 120gaatcacgcg gaaccaacgg ggcccagaca
gctagcaatg tgctgcagtg gctggccgct 180gggtttagta tcctgctgct
gatgttctac gcctatcaga cttggaagtc aacctgcggc 240tgggaggaaa
tctacgtgtg cgctattgag atggtgaaag tgatcctgga gttcttcttc
300gagttcaaga acccaagcat gctgtacctg gctactggac accgagtgca
gtggctgaga 360tatgcagaat ggctgctgac atgccccgtc atcgccattc
acctgtccaa cctgacaggc 420ctgagcaatg actactccag gagaactatg
ggactgctgg tgtccgacat cggctgcatt 480gtctggggag caacttctgc
tatggcaacc ggatacgtga aggtcatctt tttctgcctg 540gggctgtgct
atggcgcaaa tacctttttc cacgcagcca aggcctacat tgaggggtat
600cataccgtgc caaaaggccg gtgccgacag gtggtcacag gaatggcttg
gctgtttttc 660gtctcttggg gaatgtttcc catcctgttc attctggggc
ctgaagggtt cggcgtgctg 720tctgtctacg gaagtacagt ggggcatact
atcattgacc tgatgtccaa aaactgttgg 780ggcctgctgg gacactatct
gagagtgctg atccacgagc atatcctgat tcatggcgat 840attcggaaga
ccacaaaact gaatatcggc ggaaccgaga ttgaagtgga aacactggtg
900gaagacgagg ctgaggctgg ggctgtgaac aaggggactg gcaaa
94522315PRTArtificial Sequencemutated Chop2 22Met Asp Tyr Gly Gly
Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn Pro
Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln Cys Tyr
Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln Thr
Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60Leu
Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75
80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys Val Ile Leu
85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala
Thr 100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu
Leu Thr Cys 115 120 125Pro Val Ile Ala Ile His Leu Ser Asn Leu Thr
Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met Gly Leu Leu
Val Ser Asp Ile Gly Cys Ile145 150 155 160Val Trp Gly Ala Thr Ser
Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe Phe Cys Leu
Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185 190Ala Lys
Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys 195 200
205Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly
210 215 220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly
Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile
Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu Gly His
Tyr Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile His Gly
Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly Thr Glu
Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu Ala Gly
Ala Val Asn Lys Gly Thr Gly Lys305 310 31523315PRTArtificial
Sequencemutated Chop2 23Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly
Arg Glu Leu Leu Phe1 5 10 15Val Thr Asn Pro Val Val Val Asn Gly Ser
Val Leu Val Pro Glu Asp 20 25 30Gln Cys Tyr Cys Ala Gly Trp Ile Glu
Ser Arg Gly Thr Asn Gly Ala 35 40 45Gln Thr Ala Ser Asn Val Leu Gln
Trp Leu Ala Ala Gly Phe Ser Ile 50 55 60Leu Leu Leu Met Phe Tyr Ala
Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75 80Trp Glu Glu Ile Tyr
Val Cys Ala Ile Glu Met Val Lys Val Ile Leu 85 90 95Glu Phe Phe Phe
Glu Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala Thr 100 105 110Gly His
Arg Val Gln Trp Leu Arg Tyr Ala Glu Trp Leu Leu Thr Cys 115 120
125Pro Val Ile Leu Ile His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp
130 135 140Tyr Ser Arg Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly
Ala Ile145 150 155 160Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly
Tyr Val Lys Val Ile 165 170 175Phe Phe Cys Leu Gly Leu Cys Tyr Gly
Ala Asn Thr Phe Phe His Ala 180 185 190Ala Lys Ala Tyr Ile Glu Gly
Tyr His Thr Val Pro Lys Gly Arg Cys 195 200 205Arg Gln Val Val Thr
Gly Met Ala Trp Leu Phe Phe Val Ser Trp Gly 210 215 220Met Phe Pro
Ile Leu Phe Ile Leu Gly Pro Glu Gly Phe Gly Val Leu225 230 235
240Ser Val Tyr Gly Ser Thr Val Gly His Thr Ile Ile Asp Leu Met Ser
245 250 255Lys Asn Cys Trp Gly Leu Leu Gly His Tyr Leu Arg Val Leu
Ile His 260 265 270Glu His Ile Leu Ile His Gly Asp Ile Arg Lys Thr
Thr Lys Leu Asn 275 280 285Ile Gly Gly Thr Glu Ile Glu Val Glu Thr
Leu Val Glu Asp Glu Ala 290 295 300Glu Ala Gly Ala Val Asn Lys Gly
Thr Gly Lys305 310 31524945DNAArtificial Sequencemutated Chop2
24atggactacg ggggggctct gtctgctgtc gggagggaac tgctgtttgt gactaaccct
60gtcgtcgtga acgggagtgt gctggtccct gaggaccagt gctactgtgc cggctggatc
120gaatcacgcg gaaccaacgg ggcccagaca gctagcaatg tgctgcagtg
gctggccgct 180gggtttagta tcctgctgct gatgttctac gcctatcaga
cttggaagtc aacctgcggc 240tgggaggaaa tctacgtgtg cgctattgag
atggtgaaag tgatcctgga gttcttcttc 300gagttcaaga acccaagcat
gctgtacctg gctactggac accgagtgca gtggctgaga 360tatgcagaat
ggctgctgac atgccccgtc atctgcattc acctgtccaa cctgacaggc
420ctgagcaatg actactccag gagaactatg ggactgctgg tgtccgacat
cggcgccatt 480gtctggggag caacttctgc tatggcaacc ggatacgtga
aggtcatctt tttctgcctg 540gggctgtgct atggcgcaaa tacctttttc
cacgcagcca aggcctacat tgaggggtat 600cataccgtgc caaaaggccg
gtgccgacag gtggtcacag gaatggcttg gctgtttttc 660gtctcttggg
gaatgtttcc catcctgttc attctggggc ctgaagggtt cggcgtgctg
720tctgtctacg gaagtacagt ggggcatact atcattgacc tgatgtccaa
aaactgttgg 780ggcctgctgg gacactatct gagagtgctg atccacgagc
atatcctgat tcatggcgat 840attcggaaga ccacaaaact gaatatcggc
ggaaccgaga ttgaagtgga aacactggtg 900gaagacgagg ctgaggctgg
ggctgtgaac aaggggactg gcaaa 94525315PRTArtificial Sequencemutated
Chop2 25Met Asp Tyr Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu
Phe1 5 10 15Val Thr Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro
Glu Asp 20 25 30Gln Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr
Asn Gly Ala 35 40 45Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala
Gly Phe Ser Ile 50 55 60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp
Lys Ser Thr Cys Gly65 70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile
Glu Met Val Lys Val Ile Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn
Pro Ser Met Leu Tyr Leu Ala Thr 100 105 110Gly His Arg Val Gln Trp
Leu Arg Tyr Ala Glu Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Cys
Ile His Leu Ser Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser
Arg Arg Thr Met Gly Leu Leu Val Ser Asp Ile Gly Ala Ile145 150 155
160Val Trp Gly Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile
165 170 175Phe Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe
His Ala 180 185 190Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro
Lys Gly Arg Cys 195 200 205Arg Gln Val Val Thr Gly Met Ala Trp Leu
Phe Phe Val Ser Trp Gly 210 215 220Met Phe Pro Ile Leu Phe Ile Leu
Gly Pro Glu Gly Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser
Thr Val Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys
Trp Gly Leu Leu Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu
His Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280
285Ile Gly Gly Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala
290 295 300Glu Ala Gly Ala Val Asn Lys Gly Thr Gly Lys305 310
31526315PRTArtificial SequenceChR2 mutant construct 26Met Asp Tyr
Gly Gly Ala Leu Ser Ala Val Gly Arg Glu Leu Leu Phe1 5 10 15Val Thr
Asn Pro Val Val Val Asn Gly Ser Val Leu Val Pro Glu Asp 20 25 30Gln
Cys Tyr Cys Ala Gly Trp Ile Glu Ser Arg Gly Thr Asn Gly Ala 35 40
45Gln Thr Ala Ser Asn Val Leu Gln Trp Leu Ala Ala Gly Phe Ser Ile
50 55 60Leu Leu Leu Met Phe Tyr Ala Tyr Gln Thr Trp Lys Ser Thr Cys
Gly65 70 75 80Trp Glu Glu Ile Tyr Val Cys Ala Ile Glu Met Val Lys
Val Ile Leu 85 90 95Glu Phe Phe Phe Glu Phe Lys Asn Pro Ser Met Leu
Tyr Leu Ala Thr 100 105 110Gly His Arg Val Gln Trp Leu Arg Tyr Ala
Glu Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Leu Ile His Leu Ser
Asn Leu Thr Gly Leu Ser Asn Asp 130 135 140Tyr Ser Arg Arg Thr Met
Gly Leu Leu Val Ser Asp Ile Gly Thr Ile145 150 155 160Val Trp Gly
Ala Thr Ser Ala Met Ala Thr Gly Tyr Val Lys Val Ile 165 170 175Phe
Phe Cys Leu Gly Leu Cys Tyr Gly Ala Asn Thr Phe Phe His Ala 180 185
190Ala Lys Ala Tyr Ile Glu Gly Tyr His Thr Val Pro Lys Gly Arg Cys
195 200 205Arg Gln Val Val Thr Gly Met Ala Trp Leu Phe Phe Val Ser
Trp Gly 210 215 220Met Phe Pro Ile Leu Phe Ile Leu Gly Pro Glu Gly
Phe Gly Val Leu225 230 235 240Ser Val Tyr Gly Ser Thr Val Gly His
Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Asn Cys Trp Gly Leu Leu
Gly His Tyr Leu Arg Val Leu Ile His 260 265 270Glu His Ile Leu Ile
His Gly Asp Ile Arg Lys Thr Thr Lys Leu Asn 275 280 285Ile Gly Gly
Thr Glu Ile Glu Val Glu Thr Leu Val Glu Asp Glu Ala 290 295 300Glu
Ala Gly Ala Val Asn Lys Gly Thr Gly Lys305 310 315
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