U.S. patent application number 16/586524 was filed with the patent office on 2020-01-30 for dopamine receptor type 2 specific promoter and methods of use thereof.
The applicant listed for this patent is The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Karl A. Deisseroth, Charu Ramakrishnan, Kelly Zalocusky.
Application Number | 20200032291 16/586524 |
Document ID | / |
Family ID | 56092485 |
Filed Date | 2020-01-30 |
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United States Patent
Application |
20200032291 |
Kind Code |
A1 |
Deisseroth; Karl A. ; et
al. |
January 30, 2020 |
DOPAMINE RECEPTOR TYPE 2 SPECIFIC PROMOTER AND METHODS OF USE
THEREOF
Abstract
A nucleic acid containing a dopamine receptor type 2-specific
promoter (D2SP) is provided. In certain embodiments, the nucleic
acid includes a dopamine receptor type 2-specific promoter (D2SP),
wherein the D2SP does not include exon 1 of a D2 receptor gene,
wherein the D2SP comprises a Kozak sequence, and wherein the D2SP
includes a nucleotide sequence having at least 95% sequence
identity to the nucleotide sequence set forth in SEQ ID NO: 1. Also
provided are expression vectors, genetically modified host cells
and kits that include the subject nucleic acid.
Inventors: |
Deisseroth; Karl A.;
(Stanford, CA) ; Ramakrishnan; Charu; (San Jose,
CA) ; Zalocusky; Kelly; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior
University |
Stanford |
CA |
US |
|
|
Family ID: |
56092485 |
Appl. No.: |
16/586524 |
Filed: |
September 27, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15531965 |
May 31, 2017 |
10435709 |
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PCT/US15/63804 |
Dec 3, 2015 |
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16586524 |
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62087603 |
Dec 4, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2750/14143
20130101; C12N 2830/008 20130101; C12N 15/85 20130101; C07K
14/43595 20130101; C12N 9/1241 20130101; C07K 14/70571
20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; C07K 14/705 20060101 C07K014/705; C07K 14/435 20060101
C07K014/435; C12N 9/12 20060101 C12N009/12 |
Claims
1. A nucleic acid comprising a dopamine receptor type 2-specific
promoter (D2SP), wherein the D2SP does not include exon 1 of a D2
receptor gene, wherein the D2SP contains a Kozak sequence, and
wherein the D2SP contains a nucleotide sequence having at least 95%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO: 1.
2. The nucleic acid of claim 1, wherein the Kozak sequence is at
the 3' terminus of the D2SP.
3. The nucleic acid of any of claims 1 and 2, wherein the D2SP
contains a BamHI restriction site.
4. The nucleic acid of claim 3, wherein the BamHI restriction site
is located 5' of the Kozak sequence.
5. The nucleic acid of any of claims 1 to 4, wherein the D2SP
contains a nucleotide sequence having at least 98% sequence
identity to the nucleotide sequence set forth in SEQ ID NO: 1.
6. The nucleic acid of any of claims 1 to 5, wherein the D2SP is
operably linked to a nucleotide sequence encoding a gene product
that provides a detectable signal.
7. The nucleic acid of claim 6, wherein the gene product that
provides a detectable signal is a fluorescent protein.
8. The nucleic acid of claim 7, wherein the fluorescent protein is
selected from the group consisting of a green fluorescent protein,
a yellow fluorescent protein, a cyan fluorescent protein, a calcium
indicator and a voltage indicator.
9. The nucleic acid of any of claims 1 to 8, wherein the D2SP is
operably linked to a nucleotide sequence encoding a
light-responsive polypeptide.
10. The nucleic acid of claim 9, wherein the light-responsive
polypeptide is a depolarizing light-responsive polypeptide, wherein
the depolarizing light-responsive polypeptide contains an amino
acid sequence having at least 75% sequence identity to any one of
SEQ ID NOs: 4-37.
11. The nucleic acid of claim 9, wherein the light-responsive
polypeptide is a hyperpolarizing light-responsive polypeptide,
wherein the hyperpolarizing light-responsive polypeptide contains
an amino acid sequence having at least 75% sequence identity to any
one of SEQ ID NOs: 38-56.
12. The nucleic acid of any of claims 1 to 11, wherein the D2SP is
operably linked to a nucleotide sequence encoding a
recombinase.
13. The nucleic acid of claim 12, wherein the recombinase is
selected from the group consisting of a Cre recombinase and a FLP
recombinase.
14. A recombinant expression vector comprising the nucleic acid of
any of claims 1 to 13.
15. A genetically modified host cell comprising the nucleic acid of
any of claims 1 to 13, or the recombinant expression vector of
claim 14.
16. The genetically modified host cell of claim 15, wherein the
host cell is a neuronal cell.
17. The genetically modified host cell of claim 15, wherein the
host cell is a progenitor cell.
18. The genetically modified host cell of claim 17, wherein the
progenitor cell is a stem cell.
19. A method of modulating activity of a target neuron, the method
comprising introducing into the target neuron the nucleic acid of
any of claims 1 to 13, wherein the D2SP is operably linked to a
light-responsive polypeptide that, when activated by light, induces
hyperpolarization or depolarization of the target neuron.
20. A method of fluorescently labeling a target cell, the method
comprising introducing into the target cell the nucleic acid of any
of claims 1 to 13, wherein the D2SP is operably linked to a
fluorescent protein that, when expressed, fluorescently labels the
target cell.
21. The method of claim 20, wherein the target cell is a neuronal
cell.
22. The method of claim 20, wherein the target cell is a progenitor
cell.
23. The method of claim 22, wherein the progenitor cell is a stem
cell.
24. A kit comprising: a recombinant expression vector that
comprises a nucleic acid comprising a dopamine receptor type
2-specific promoter (D2SP), wherein the D2SP does not include exon
1 of a D2 receptor gene, wherein the D2SP contains a Kozak
sequence, and wherein the D2SP comprises a nucleotide sequence
having at least 95% sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 1; and instructions for introducing the
recombinant expression vector into a target cell.
25. The kit of claim 24, wherein the kit further comprises a
control expression vector that contains a nucleic acid containing a
dopamine receptor type 2-specific promoter (D2SP), wherein the D2SP
does not include exon 1 of a D2 receptor gene, wherein the D2SP
comprises a Kozak sequence, and wherein the D2SP comprises a
nucleotide sequence having at least 95% sequence identity to the
nucleotide sequence set forth in SEQ ID NO: 1.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 62/087,603, filed Dec. 4, 2014, which
application is incorporated herein by reference in its
entirety.
INTRODUCTION
[0002] Dopamine is a catecholamine neurotransmitter involved in
signaling between cells in the brain and throughout the body.
Dopamine exerts its cellular and biochemical effects on target
cells by binding to its receptor, a G protein-coupled,
seven-transmembrane receptor. The dopamine type 2 (D2) receptor is
one of several dopamine receptors that have been identified. Cells,
including neurons, which express the D2 receptor, are involved in
many psychological disorders, including drug addiction, obesity,
and gambling disorders.
SUMMARY
[0003] A nucleic acid comprising a dopamine receptor type
2-specific promoter (D2SP) is provided. In certain embodiments, the
nucleic acid comprises a dopamine receptor type 2-specific promoter
(D2SP), wherein the D2SP does not include exon 1 of a D2 receptor
gene, wherein the D2SP comprises a Kozak sequence, and wherein the
D2SP includes a nucleotide sequence having at least 95% sequence
identity to the nucleotide sequence set forth in SEQ ID NO: 1. In
some cases, the Kozak sequence is at the 3' terminus of the D2SP.
In some cases, the D2SP includes a BamHI restriction site. In
certain embodiments, the BamHI restriction site is located 5' of
the Kozak sequence. In some cases, the D2SP comprises a nucleotide
sequence having at least 98% sequence identity to the nucleotide
sequence set forth in SEQ ID NO: 1.
[0004] In any embodiment set out above or infra, the D2SP is
operably linked to a nucleotide sequence encoding a gene product
that provides a detectable signal. In certain embodiments, the gene
product that provides a detectable signal is a fluorescent protein.
In some embodiments, the fluorescent protein is selected from the
group consisting of a green fluorescent protein, a yellow
fluorescent protein, a cyan fluorescent protein, a calcium
indicator and a voltage indicator.
[0005] In any embodiment set out above or infra, the D2SP is
operably linked to a nucleotide sequence encoding a
light-responsive polypeptide. In certain embodiments, the
light-responsive polypeptide is a depolarizing light-responsive
polypeptide, wherein the depolarizing light-responsive polypeptide
includes an amino acid sequence having at least 75% sequence
identity to any one of SEQ ID NOs: 4-23. In some embodiments, the
light-responsive polypeptide is a hyperpolarizing light-responsive
polypeptide, wherein the hyperpolarizing light-responsive
polypeptide includes an amino acid sequence having at least 75%
sequence identity to any one of SEQ ID NOs: 24-54.
[0006] In any embodiment set out above or infra, the D2SP is
operably linked to a nucleotide sequence encoding a recombinase. In
certain embodiments, the recombinase is selected from the group
consisting of a Cre recombinase and a FLP recombinase.
[0007] Also provided herein is a recombinant expression vector
comprising the nucleic acid of any of the above embodiments.
[0008] Also provided herein is a genetically modified host cell
comprising the nucleic acid of any of the above nucleic acid
embodiments, or the recombinant expression vector of any of the
above expression vector embodiments. In certain embodiments, the
host cell is a neuronal cell. In certain embodiments, the host cell
is a progenitor cell. In certain embodiments, the progenitor cell
is a stem cell.
[0009] Also provided herein is a method of modulating activity of a
target neuron, the method comprising introducing into the target
neuron the nucleic acid of any of the above nucleic acid
embodiments, wherein the D2SP is operably linked to a
light-responsive polypeptide that, when activated by light, induces
hyperpolarization or depolarization of the target neuron.
[0010] Also provided herein is a method of fluorescently labeling a
target cell, the method comprising introducing into the target cell
the nucleic acid of any of any of the above embodiments, wherein
the D2SP is operably linked to a fluorescent protein that, when
expressed, fluorescently labels the target cell. In certain
embodiments, the target cell is a neuronal cell. In certain
embodiments, the target cell is a progenitor cell. In certain
embodiments, the progenitor cell is a stem cell.
[0011] Also provided herein is a kit comprising: a) a recombinant
expression vector that comprises a nucleic acid comprising a
dopamine receptor type 2-specific promoter (D2SP), wherein the D2SP
does not include exon 1 of a D2 receptor gene, wherein the D2SP
includes a Kozak sequence, and wherein the D2SP comprises a
nucleotide sequence having at least 95% sequence identity to the
nucleotide sequence set forth in SEQ ID NO: 1; and b) instructions
for introducing the recombinant expression vector into a target
cell.
[0012] In any of the kit embodiments described above or infra, the
kit further comprises a control expression vector that comprises a
nucleic acid comprising a dopamine receptor type 2-specific
promoter (D2SP), wherein the D2SP does not include exon 1 of a D2
receptor gene, wherein the D2SP includes a Kozak sequence, and
wherein the D2SP comprises a nucleotide sequence having at least
95% sequence identity to the nucleotide sequence set forth in SEQ
ID NO: 1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows a schematic drawing of the conventional
promoter for the type 2 dopamine receptor (D2R) and the D2 specific
promoter (D2SP), according to an embodiment of the present
disclosure.
[0014] FIG. 2 shows aligned nucleotide sequences of D2SP (SEQ ID
NO: 1) and the conventional D2 receptor promoter (D2R; SEQ ID NO:
2), according to an embodiment of the present disclosure.
[0015] FIG. 3 shows staining of tissue slices with a D2
receptor-specific antibody using a standard protocol (left) and a
modified protocol (right).
[0016] FIG. 4 shows rat hippocampal primary neurons expressing
eNpHR 3.0-EYFP under the D2SP and antibody stained for D2 receptors
using the modified staining protocol, according to an embodiment of
the present disclosure.
[0017] FIG. 5 shows expression of eNpHR 3.0-EYFP under the D2SP and
under the conventional D2 receptor promoter (D2R) and antibody
staining for D2 receptors, according to an embodiment of the
present disclosure.
[0018] FIGS. 6-14 show schematic maps of recombinant expression
vectors containing a D2SP, according to an embodiment of the
present disclosure.
[0019] FIG. 15 shows a nucleotide sequence of exon 1 of the rat D2
receptor.
[0020] FIG. 16 shows the amino acid sequences of depolarizing
light-responsive polypeptides and derivatives thereof (SEQ ID NOs:
4-23), according to an embodiment of the present disclosure.
[0021] FIG. 17 shows the amino acid sequences of hyperpolarizing
light-responsive polypeptides and derivatives thereof (SEQ ID NOs:
24-54), according to an embodiment of the present disclosure.
[0022] FIG. 18 shows the peptide sequences (SEQ ID NOs: 55-66) that
may be used to enhance expression of the light-responsive
polypeptides in a host cell or a target cell, according to an
embodiment of the present disclosure.
DEFINITIONS
[0023] The terms "polynucleotide", "nucleotide", "nucleotide
sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid
sequence" and "oligonucleotide" are used interchangeably, and can
also include plurals of each respectively depending on the context
in which the terms are utilized. They refer to a polymeric form of
nucleotides of any length, either deoxyribonucleotides (DNA) or
ribonucleotides (RNA), or analogs thereof. Polynucleotides may have
any three-dimensional structure, and may perform any function,
known or unknown. The following are non-limiting examples of
polynucleotides: coding or non-coding regions of a gene or gene
fragment, loci (locus) defined from linkage analysis, exons,
introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA,
ribozymes, small interfering RNA, (siRNA), microRNA (miRNA), small
nuclear RNA (snRNA), cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA (A, B and Z
structures) of any sequence, PNA, locked nucleic acid (LNA), TNA
(treose nucleic acid), isolated RNA of any sequence, nucleic acid
probes, and primers. LNA, often referred to as inaccessible RNA, is
a modified RNA nucleotide. The ribose moiety of an LNA nucleotide
is modified with an extra bridge connecting the 2' and 4' carbons.
The bridge "locks" the ribose in the 3'-endo structural
conformation, which is often found in the A-form of DNA or RNA,
which can significantly improve thermal stability.
[0024] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The polymer may be linear, it may comprise modified amino
acids, and it may be interrupted by non-amino acids. The terms also
encompass an amino acid polymer that has been modified; for
example, disulfide bond formation, glycosylation, lipidation,
acetylation, phosphorylation, or any other manipulation, such as
conjugation with a labeling component. As used herein the term
"amino acid" refers to either natural and/or unnatural or synthetic
amino acids, including glycine and both the D or L optical isomers,
and amino acid analogs and peptidomimetics.
[0025] As used herein, "sequence identity" or "identity" in the
context of two nucleic acid sequences makes reference to a
specified percentage of residues in the two sequences that are the
same when aligned for maximum correspondence over a specified
comparison window, as measured by sequence comparison algorithms or
by visual inspection. When percentage of sequence identity is used
in reference to proteins it is recognized that residue positions
which are not identical often differ by conservative amino acid
substitutions, where amino acid residues are substituted for other
amino acid residues with similar chemical properties (e.g., charge
or hydrophobicity) and, therefore, do not change the functional
properties of the molecule. When sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences that differ by such conservative substitutions are said
to have "sequence similarity" or "similarity." Any suitable means
for making this adjustment may be used. This may involve scoring a
conservative substitution as a partial rather than a full mismatch,
thereby increasing the percentage sequence identity. Thus, for
example, where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1. The
scoring of conservative substitutions is calculated, e.g., as
implemented in the program PC/GENE (Intelligenetics, Mountain View,
Calif.).
[0026] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may include additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not include additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0027] Any suitable methods of alignment of sequences for
comparison may be employed. Thus, the determination of percent
identity between any two sequences can be accomplished using a
mathematical algorithm. Preferred, non-limiting examples of such
mathematical algorithms are the algorithm of Myers and Miller,
CABIOS, 4:11 (1988), which is hereby incorporated by reference in
its entirety; the local homology algorithm of Smith et al, Adv.
Appl. Math., 2:482 (1981), which is hereby incorporated by
reference in its entirety; the homology alignment algorithm of
Needleman and Wunsch, JMB, 48:443 (1970), which is hereby
incorporated by reference in its entirety; the
search-for-similarity-method of Pearson and Lipman, Proc. Natl.
Acad. Sci. USA, 85:2444 (1988), which is hereby incorporated by
reference in its entirety; the algorithm of Karlin and Altschul,
Proc. Natl. Acad. Sci. USA, 87:2264 (1990), which is hereby
incorporated by reference in its entirety; modified as in Karhn and
Altschul, Proc. Natl. Acad. Sci. USA, 90:5873 (1993), which is
hereby incorporated by reference in its entirety.
[0028] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity. Such implementations include, but are not limited to:
CLUSTAL in the PC/Gene program (available from Intelligenetics,
Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP,
BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics
Software Package, Version 8 (available from Genetics Computer Group
(GCG), 575 Science Drive, Madison, Wis., USA). Alignments using
these programs can be performed using the default parameters. The
CLUSTAL program is well described by Higgins et al., Gene, 73:237
(1988), Higgins et al., CABIOS, 5:151 (1989); Corpet et al., Nucl.
Acids Res., 16:10881 (1988); Huang et al., CABIOS, 8:155 (1992);
and Pearson et al., Meth. Mol. Biol., 24:307 (1994), which are
hereby incorporated by reference in their entirety. The ALIGN
program is based on the algorithm of Myers and Miller, supra. The
BLAST programs of Altschul et al., JMB, 215:403 (1990); Nucl. Acids
Res., 25:3389 (1990), which are hereby incorporated by reference in
their entirety, are based on the algorithm of Karlin and Altschul
supra.
[0029] Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information (NCBI;
worldwideweb.ncbi.nlm.nih.gov).
[0030] As used herein, "expression" refers to the process by which
a polynucleotide is transcribed into mRNA and/or the process by
which the transcribed mRNA (also referred to as "transcript") is
subsequently being translated into peptides, polypeptides, or
proteins. The transcripts and the encoded polypeptides are
collectedly referred to as "gene product," depending on the
context.
[0031] "Gene" refers to a polynucleotide sequence that includes
control and coding sequences necessary for the production of a
polypeptide or precursor. The polypeptide can be encoded by a full
length coding sequence or by any portion of the coding sequence. A
gene may constitute an uninterrupted coding sequence or it may
include one or more introns, bound by the appropriate splice
junctions. Moreover, a gene may comprise one or more modifications
in either the coding or the untranslated regions that could affect
the biological activity or the chemical structure of the expression
product, the rate of expression, or the manner of expression
control. Such modifications include, but are not limited to,
mutations, insertions, deletions, and substitutions of one or more
nucleotides. In this regard, such modified genes may be referred to
as "variants" of the "native" gene.
[0032] The term "genetic modification" refers to a permanent or
transient genetic change induced in a cell following introduction
into the cell of a heterologous nucleic acid (i.e., nucleic acid
exogenous to the cell). Genetic change ("modification") can be
accomplished by incorporation of the heterologous nucleic acid into
the genome of the host cell, or by transient or stable maintenance
of the heterologous nucleic acid as an extrachromosomal element.
Where the cell is a eukaryotic cell, a permanent genetic change can
be achieved by introduction of the nucleic acid into the genome of
the cell. Suitable methods of genetic modification include viral
infection, transfection, conjugation, protoplast fusion,
electroporation, particle gun technology, calcium phosphate
precipitation, direct microinjection, and the like.
[0033] The term "promoter" as used herein refers to a sequence of
DNA that directs the expression (transcription) of a gene. A
promoter may direct the transcription of a prokaryotic or
eukaryotic gene. A promoter may be "inducible", initiating
transcription in response to an inducing agent or, in contrast, a
promoter may be "constitutive", whereby an inducing agent does not
regulate the rate of transcription. A promoter may be regulated in
a tissue-specific or tissue-preferred manner, such that it is only
active in transcribing the operable linked coding region in a
specific tissue type or types.
[0034] The term "operably-linked" refers to a functional linkage
between a regulatory sequence and a coding sequence. The components
so described are thus in a relationship permitting them to function
in their intended manner. For example, placing a coding sequence
under regulatory control of a promoter means positioning the coding
sequence such that the expression of the coding sequence is
controlled by the promoter.
[0035] As used herein, "terminus," or "end" with respect to the
terminus or end of a nucleotide or amino acid sequence, refers to
the 5' or 3' end of a nucleotide sequence, or the amino or carboxyl
end of an amino acid sequence. Thus, a sequence at the terminus of
a nucleotide sequence or polypeptide sequence is a sequence that
includes the 5'-most or 3'-most nucleotide of the nucleotide
sequence, or the amino or carboxyl end of the polypeptide
sequence.
[0036] The terms "light-activated," "light-responsive" in reference
to a polypeptide or protein that is light-responsive, are used
interchangeably and include light-responsive ion channels or
opsins, and ion pumps as described herein. Such light-responsive
proteins may have a depolarizing or hyperpolarizing effect on the
cell on whose plasma membrane the protein is expressed depending on
the ion permeability of the activated protein, and the
electrochemical gradients present across the plasma membrane.
[0037] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0038] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0039] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0040] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a genetically modified host cell" includes a
plurality of such genetically modified host cells and reference to
"the neuronal cell" includes reference to one or more neuronal
cells and equivalents thereof known to those skilled in the art,
and so forth. It is further noted that the claims may be drafted to
exclude any optional element. As such, this statement is intended
to serve as antecedent basis for use of such exclusive terminology
as "solely," "only" and the like in connection with the recitation
of claim elements, or use of a "negative" limitation.
[0041] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination.
All combinations of the embodiments pertaining to the invention are
specifically embraced by the present invention and are disclosed
herein just as if each and every combination was individually and
explicitly disclosed. In addition, all sub-combinations of the
various embodiments and elements thereof are also specifically
embraced by the present invention and are disclosed herein just as
if each and every such sub-combination was individually and
explicitly disclosed herein.
[0042] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
DETAILED DESCRIPTION
[0043] A nucleic acid comprising a dopamine receptor type
2-specific promoter (D2SP) and methods of using the same to express
a polypeptide in a target cell of interest are provided. Aspects of
the present disclosure include a nucleic acid comprising a D2SP
wherein the D2SP does not include exon 1 of a D2 receptor gene,
wherein the D2SP comprises a Kozak sequence, and wherein the D2SP
comprises a nucleotide sequence having at least 95% sequence
identity to the nucleotide sequence set forth in SEQ ID NO: 1 (FIG.
2).
[0044] In some embodiments, a subject nucleic acid comprises a
D2SP, wherein the D2SP does not include exon 1 of a D2 receptor
gene, wherein the D2SP comprises a Kozak sequence, and wherein the
D2SP comprises a nucleotide sequence having at least 75%, e.g., at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity to the nucleotide sequence set forth in SEQ
ID NO: 1.
[0045] In certain embodiments, a subject nucleic acid comprises a
D2SP wherein the D2SP does not include exon 1 of a D2 receptor
gene, wherein the D2SP comprises a Kozak sequence at the 3'
terminus of the D2SP, wherein the D2SP comprises a BamHI
restriction site located 5' of the Kozak sequence, and wherein the
D2SP comprises a nucleotide sequence having at least 75%, e.g., at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity to the nucleotide sequence set forth in SEQ
ID NO: 1.
[0046] In certain embodiments, a subject nucleic acid comprises a
D2SP wherein the D2SP does not include exon 1 of a D2 receptor
gene, wherein the D2SP comprises a Kozak sequence at the 3'
terminus of the D2SP, wherein the D2SP comprises a BamHI
restriction site located 5' of the Kozak sequence, wherein the D2SP
comprises a nucleotide sequence having at least 75%, e.g., at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO: 1, and wherein the D2SP is operably linked to a nucleotide
sequence encoding a gene product. In some cases, the gene product
is a polypeptide. In some cases, the gene product is a
polynucleotide. In some instances, the gene product is a
polypeptide that provides a detectable signal, such as a
fluorescent protein; a genetically encoded indicator; a
light-responsive polypeptide; a recombinase; or a combination
thereof.
[0047] In certain embodiments, the subject nucleic acid comprises a
D2SP wherein the D2SP does not include exon 1 of a D2 receptor
gene, wherein the D2SP comprises a Kozak sequence at the 3'
terminus of the D2SP, wherein the D2SP comprises a BamHI
restriction site located 5' of the Kozak sequence, wherein the D2SP
comprises a nucleotide sequence having at least 75%, e.g., at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO: 1, and wherein the D2SP is operably linked to a nucleotide
sequence encoding a light-responsive polypeptide selected from the
polypeptides of SEQ ID NOs: 4-54.
[0048] In certain embodiments, the subject nucleic acid comprises a
D2SP wherein the D2SP does not include exon 1 of a D2 receptor
gene, wherein the D2SP comprises a Kozak sequence at the 3'
terminus of the D2SP, wherein the D2SP comprises a BamHI
restriction site located 5' of the Kozak sequence, wherein the D2SP
comprises a nucleotide sequence having at least 75%, e.g., at least
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to the nucleotide sequence set forth in SEQ ID
NO: 1, and wherein the D2SP is operably linked to a nucleotide
sequence encoding a fluorescent protein selected from a green
fluorescent protein, a yellow fluorescent protein, a cyan
fluorescent protein, a calcium indicator and a voltage
indicator.
[0049] Also provided herein is a recombinant expression vector
comprising a nucleic acid that includes a D2SP, wherein the D2SP
does not include exon 1 of a D2 receptor gene, wherein the D2SP
comprises a Kozak sequence, and wherein the D2SP comprises a
nucleotide sequence having at least 75%, e.g., at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to the nucleotide sequence set forth in SEQ ID NO: 1.
[0050] In certain embodiments, the recombinant expression vector
comprises a nucleic acid that includes a D2SP, wherein the D2SP
does not include exon 1 of a D2 receptor gene, wherein the D2SP
comprises a Kozak sequence at the 3' terminus of the D2SP, wherein
the D2SP comprises a BamHI restriction site located 5' of the Kozak
sequence, wherein the D2SP comprises a nucleotide sequence having
at least 75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide
sequence set forth in SEQ ID NO: 1, and wherein the D2SP is
operably linked to a nucleotide sequence encoding a gene product.
In some instances, the gene product is a polypeptide that provides
a detectable signal, such as a fluorescent protein; a genetically
encoded indicator; a light-responsive polypeptide; a recombinase;
or a combination thereof.
[0051] Also provided herein is a genetically modified host cell
comprising a nucleic acid that comprises a D2SP, wherein the D2SP
does not include exon 1 of a D2 receptor gene, wherein the D2SP
comprises a Kozak sequence, and wherein the D2SP comprises a
nucleotide sequence having at least 75%, e.g., at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to the nucleotide sequence set forth in SEQ ID NO: 1. In
some instances the nucleic acid is contained in a recombinant
expression vector in the genetically modified host cell.
[0052] In certain embodiments, a genetically modified host cell of
the present disclosure comprises a recombinant expression vector
comprising a nucleic acid that comprises a D2SP, wherein the D2SP
does not include exon 1 of a D2 receptor gene, wherein the D2SP
comprises a Kozak sequence at the 3' terminus of the D2SP, wherein
the D2SP comprises a BamHI restriction site located 5' of the Kozak
sequence, wherein the D2SP comprises a nucleotide sequence having
at least 75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide
sequence set forth in SEQ ID NO: 1, and wherein the D2SP is
operably linked to a nucleotide sequence encoding a gene product.
In some instances, the gene product is a polypeptide that provides
a detectable signal, such as a fluorescent protein; a genetically
encoded indicator; a light-responsive polypeptide; a recombinase;
or a combination thereof.
[0053] Also provided herein is a method of modulating activity of a
target neuron, the method including introducing into the target
neuron a nucleic acid that comprises a D2SP wherein the D2SP does
not include exon 1 of a D2 receptor gene, wherein the D2SP
comprises a Kozak sequence at the 3' terminus of the D2SP and a
BamHI restriction site located 5' of the Kozak sequence, and
wherein the D2SP comprises a nucleotide sequence having at least
75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 1, wherein the D2SP is operably linked to a
light-responsive polypeptide that, when activated by light, induces
hyperpolarization or depolarization of the target neuron.
[0054] Also provided herein is a method of modulating activity of a
target neuron, the method comprising introducing into the target
neuron a nucleic acid that comprises a D2SP wherein the D2SP does
not include exon 1 of a D2 receptor gene, wherein the D2SP
comprises a Kozak sequence at the 3' terminus of the D2SP and a
BamHI restriction site located 5' of the Kozak sequence, and
wherein the D2SP comprises a nucleotide sequence having at least
75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 1, wherein the D2SP is operably linked to a
light-responsive polypeptide comprising the amino acid sequence set
forth in any one of SEQ ID NOs: 4-23, that, when activated by
light, induces depolarization of the target neuron.
[0055] Also provided herein is a method of modulating activity of a
target neuron, the method comprising introducing into the target
neuron a nucleic acid that comprises a D2SP wherein the D2SP does
not include exon 1 of a D2 receptor gene, wherein the D2SP
comprises a Kozak sequence at the 3' terminus of the D2SP and a
BamHI restriction site located 5' of the Kozak sequence, and
wherein the D2SP comprises a nucleotide sequence having at least
75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 1, wherein the D2SP is operably linked to a
light-responsive polypeptide comprising the amino acid sequence set
forth in any one of SEQ ID NOs: 24-54, that, when activated by
light, induces hyperpolarization of the target neuron.
[0056] Also provided herein is a method of fluorescently labeling a
target cell, the method comprising introducing into the target cell
a nucleic acid that comprises a D2SP wherein the D2SP does not
include exon 1 of a D2 receptor gene, wherein the D2SP comprises a
Kozak sequence at the 3' terminus of the D2SP and a BamHI
restriction site located 5' of the Kozak sequence, and wherein the
D2SP comprises a nucleotide sequence having at least 75%, e.g., at
least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity to the nucleotide sequence set forth in SEQ
ID NO: 1, wherein the D2SP is operably linked to a fluorescent
protein that, when expressed, fluorescently labels the target
cell.
[0057] In certain embodiments, a method of the present disclosure
of fluorescently labeling a target cell comprises introducing into
a target neuron a nucleic acid that comprises a D2SP wherein the
D2SP does not include exon 1 of a D2 receptor gene, wherein the
D2SP comprises a Kozak sequence at the 3' terminus of the D2SP and
a BamHI restriction site located 5' of the Kozak sequence, and
wherein the D2SP comprises a nucleotide sequence having at least
75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 1, wherein the D2SP is operably linked to a
fluorescent protein that, when expressed, fluorescently labels the
target neuron.
[0058] In certain embodiments, a method of the present disclosure
of fluorescently labeling a target cell comprises introducing into
a target progenitor cell a nucleic acid that includes a D2SP
wherein the D2SP does not include exon 1 of a D2 receptor gene,
wherein the D2SP comprises a Kozak sequence at the 3' terminus of
the D2SP and a BamHI restriction site located 5' of the Kozak
sequence, and wherein the D2SP comprises a nucleotide sequence
having at least 75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the
nucleotide sequence set forth in SEQ ID NO: 1, wherein the D2SP is
operably linked to a fluorescent protein that, when expressed,
fluorescently labels the target progenitor cell.
[0059] In certain embodiments, a method of the present disclosure
of fluorescently labeling a target cell comprises introducing into
a target stem cell a nucleic acid that comprises a D2SP wherein the
D2SP does not include exon 1 of a D2 receptor gene, wherein the
D2SP comprises a Kozak sequence at the 3' terminus of the D2SP and
a BamHI restriction site located 5' of the Kozak sequence, and
wherein the D2SP comprises a nucleotide sequence having at least
75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 1, wherein the D2SP is operably linked to a
fluorescent protein that, when expressed, fluorescently labels the
target stem cell.
[0060] Further aspects of the present disclosure will now be
described in more detail below.
Nucleic Acids
Dopamine Receptor Type 2-Specific Promoter (D2SP)
[0061] As summarized above, aspects of the present disclosure
include a nucleic acid comprising a D2SP, i.e., a promoter sequence
that directs expression of genes operably linked to the promoter in
cells that express the type 2 dopamine (D2) receptor. In certain
embodiments, the D2SP is derived from a genomic sequence 5' of the
first exon of a D2 receptor in a genome.
[0062] In certain embodiments, the D2 receptor is derived from a
mammalian genome, such as, but not limited to, rat, mouse, monkey,
non-human primate or human genome.
[0063] In some embodiments, the D2SP is derived from a genomic
sequence that is 5' of the first exon of a D2 receptor. Thus, in
some embodiments, the D2SP is derived from a genomic sequence that
begins 3.0 kilobases (kb) or less, e.g., 2.5 kb of less, such as
2.0 kb or less, including 1.6 kb or less 5' of the beginning of the
first exon of a D2 receptor. In other embodiments, the D2SP is
derived from a genomic sequence that begins 0.5 kilobases (kb) or
more, e.g., 1.0 kb of more, such as 1.2 kb or more, including 1.5
kb or more 5' of the beginning of the first exon of a D2 receptor.
In certain embodiments, the D2SP is derived from a genomic sequence
that begins in the range of 3.0 to 0.5 kb, e.g., 2.5 kb to 1.0 kb,
or 2.0 kb to 1.2 kb 5' of the beginning of the first exon or
transcriptional start site of the D2 receptor.
[0064] The transcriptional start site, or the beginning of the
first exon of a gene, as used interchangeably herein, may be
defined as the 5' end of a mature RNA (mRNA) transcribed from the
genetic locus encoding the gene. Thus in certain embodiments, the
beginning of the first exon of a D2 receptor is defined by the 5'
end of the mRNA transcribed from the D2 receptor genomic locus. In
certain embodiments, the beginning of the first exon of a D2
receptor is defined by the sequence represented by GenBank
Accession numbers: NM_012547 (Rattus norvegicus); NM_010077 (Mus
musculus); or NM_000795 (Homo sapiens).
[0065] In certain embodiments, the length of the D2SR is from 500
base pairs (bp) to 2500 bp, e.g., 750 bp to 2250 bp, 1000 bp to
2000 bp, including 1250 bp to 1750 bp. In some instances, the
length of the D2SR is 500 bp or more, e.g., 750 bp or more, 1000 bp
or more, 1250 bp or more, 1350 bp or more, 1450 bp or more, 1500 bp
or more, 1510 bp or more, 1520 bp or more, or 1530 bp or more. In
some instances, the length of the D2SR is 2000 bp or less, e.g.,
1750 bp or less, 1700 bp or less, 1650 bp or less, 1600 bp or less,
1590 bp or less, 1580 bp or less, 1570 bp or less, 1560 bp or less,
or 1550 bp or less. In another embodiment, the length of the D2SR
is about 1540 bp.
[0066] Aspects of the present disclosure include a nucleic acid
that comprises a D2SP that does not include exon 1 of a D2 receptor
gene (FIG. 1). The D2 receptor gene may be any mammalian D2
receptor gene, including, but not limited to the rat D2 receptor
gene (GeneID 24318), the mouse D2 receptor gene (GeneID 13489) or
the human D2 receptor gene (GeneID 1813). Other mammalian D2
receptor genes include monkey and non-human primate D2 receptor
genes. Any suitable method for determining the first exon of a D2
receptor gene may be used. In certain embodiments, the exon 1 of a
rat D2 receptor gene is defined by the sequence that is 80% or
more, e.g., 85% or more, 90% or more, 95% or more, 98% or more, 99%
or more, or 100% identical to the sequence shown in SEQ ID NO: 3
(FIG. 15). Thus, in certain embodiments, the D2SP does not include
a sequence that is 80% or more, e.g., 85% or more, 90% or more, 95%
or more, 98% or more, 99% or more, or 100% identical to the
sequence shown in SEQ ID NO: 3 (FIG. 15). In certain embodiments,
the D2SP does not include a nucleotide sequence that is 90% or
more, e.g., 95% or more, 98% or more, 99% or more, or 100%
identical to nucleotides 1-313, e.g., nucleotides 1-300,
nucleotides 1-250, nucleotides 1-200, nucleotides 1-150,
nucleotides 1-100, nucleotides 1-90, nucleotides 1-80, nucleotides
1-70, nucleotides 1-60, nucleotides 1-50, nucleotides 1-40,
including nucleotides 1-30, of the sequence shown in SEQ ID NO: 3
(FIG. 15). In certain embodiments, the D2SP does not include a
nucleotide sequence that is 90% or more, e.g., 95% or more, 98% or
more, 99% or more, or 100% identical to nucleotides 1-270 of the
sequence shown in SEQ ID NO: 3 (FIG. 15).
[0067] Further aspects of the present disclosure include a nucleic
acid that comprises a D2SP comprising a Kozak sequence. The term
"Kozak sequence" refers to a sequence for facilitating the initial
binding of mRNA to the small subunit of the ribosome for initiation
of translation. An exemplary Kozak sequence is GCCRCC where R is a
purine (A or G). In certain embodiments, the Kozak sequence is
GCCACC. In certain embodiments, one, two, three or more nucleotides
may be substituted in the exemplary Kozak sequence without
significantly affecting the ability of the Kozak sequence to
function. (Kozak, M., Cell, 44(2):283-92, 1986; Kozak, M. Nucleic
Acids Res., October 26; 15(20):8125-48, 1987; Kozak, M, J. Biol.
Chem., 266(30): 19867-19870, 1991.)
[0068] In certain embodiments, the Kozak sequence is at the 3'
terminus, or end, of the D2SP. Thus, in certain embodiments where
the D2SP directs expression of an RNA transcript encoding a
polypeptide, the coding sequence for the polypeptide starts
immediately 3' of the terminal end of the D2SP. "Immediately," as
used herein in reference to a first sequence that is immediately
adjacent to a second sequence, indicates that there are no
intervening sequences (i.e., no nucleotides or amino acids) between
the first and second sequences. Thus, in certain embodiments, the
Kozak sequence is immediately followed 3' by the start codon (i.e.,
the nucleotide sequence ATG) of the coding sequence.
[0069] In certain embodiments, the D2SP includes a recognition site
for a restriction nuclease. In certain embodiments, the restriction
nuclease is BamHI. The recognition site of BamHI is GGATCC. Thus,
in certain embodiments, the D2SP includes a BamHI recognition site,
defined by the sequence GGATCC. In certain embodiments, the BamHI
restriction site is located 5' of the Kozak sequence. In certain
embodiments, the BamHI site is located immediately 5' of the Kozak
sequence. In some embodiments, the Bam HI restriction site is
located 3' of the genomic sequence of the D2 receptor genomic locus
from which the D2SP is derived. Thus, in certain embodiments, the
Bam HI restriction site is located 3' of the genomic sequence of
the D2 receptor genomic locus from which the D2SP is derived and 5'
of the Kozak sequence.
[0070] In certain embodiments, the D2SP includes a nucleotide
sequence having at least 75%, e.g., at least 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to
the nucleotide sequence set forth in SEQ ID NO: 1 (FIG. 2). Thus,
an aspect of the present disclosure includes a nucleic acid
comprising a D2SP, wherein the D2SP does not include exon 1 of a D2
receptor gene, wherein the D2SP comprises a Kozak sequence, and
wherein the D2SP comprises a nucleotide sequence having at least
75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 1. In another aspect, a subject nucleic
acid comprises a D2SP, wherein the D2SP does not include exon 1 of
a D2 receptor gene, wherein the D2SP comprises a Kozak sequence and
a BamHI restriction site, and wherein the D2SP comprises a
nucleotide sequence having at least 75%, e.g., at least 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to the nucleotide sequence set forth in SEQ ID NO: 1. In
certain embodiments, the nucleic acid comprises a D2SP, wherein the
D2SP does not include exon 1 of a D2 receptor gene, wherein the
D2SP comprises a Kozak sequence at the 3' terminus of the D2SP and
a BamHI restriction site located 5' of the Kozak sequence, and
wherein the D2SP comprises a nucleotide sequence having at least
75%, e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% sequence identity to the nucleotide sequence
set forth in SEQ ID NO: 1.
[0071] In certain embodiments, the D2SP is operably linked to a
nucleotide sequence encoding a gene product. The gene product may
be any suitable gene product that finds use being expressed
specifically in target cells that express the D2 receptor. In some
cases, the gene product is a polypeptide. In some cases, the gene
product is a polynucleotide. In certain embodiments, the gene
product is a light-responsive polypeptide. In certain embodiments,
the light-responsive polypeptide is a polypeptide that, when
expressed on the cell membrane of the target cell and activated by
exposure to light of appropriate wavelength and intensity,
depolarizes or activates the target cell. In certain embodiments,
the light-responsive polypeptide is a polypeptide that, when
expressed on the cell membrane of the target cell and activated by
exposure to light of appropriate wavelength and intensity,
hyperpolarizes or inhibits the target cell. Exemplary
light-responsive polypeptides that may be operably linked to the
subject D2SP are further described below.
[0072] In certain embodiments, the gene product operably linked to
a D2SP provides a detectable signal. A detectable signal may be
fluorescence, chemiluminescence, enzymatic activity, etc. In
certain embodiments, the gene product to which the D2SP is operably
linked and that provides a detectable signal is a fluorescent
protein, including, but not limited to, a green fluorescent
protein, a yellow fluorescent protein, a cyan fluorescent protein,
etc. In some embodiments, the gene product to which the D2SP is
operably linked and that provides a detectable signal is a
genetically encoded indicator, such as, but not limited to, a
calcium indicator or a voltage indicator. A calcium indicator is a
fluorescent polypeptide that is engineered to bind one or more
calcium ions, wherein the binding of the calcium ions alters the
fluorescence properties, such as intensity, excitation and/or
emission wavelengths, etc., of the polypeptide. Any suitable
calcium indicator may be used to provide a detectable signal in the
target cell. In some instances, the calcium indicator is a
ratiometric calcium indicator, such as Cameleon and derivatives
thereof. Other calcium indicators of interest include, but are not
limited to GCaMP1, GCaMP2, GCaMP3, and derivatives thereof, as well
as those cited in U.S. Pat. No. 8,629,256, and Tian et al. 2012
Prog Brain Res, 196:79 which are incorporated herein by reference.
A voltage indicator is a fluorescent polypeptide that is engineered
to respond to changes in membrane potential, wherein a change in
membrane potential alters the fluorescence properties, such as
intensity, excitation and/or emission wavelengths, etc., of the
polypeptide. Any suitable voltage indicator may be used to provide
a detectable signal in the target cell. Voltage indicators of
interest include, but are not limited to QuasAr1, QuasAr2, VSFP,
and derivatives thereof, as well as those cited in US App. Pub. No.
20130224756, Hochbaum et al., Nat Methods 2014 11:825, Baker et al.
Brain Cell Biol 2008 36:53; and Mutoh et al., Exp Physiol 2011
96:13, which are incorporated herein by reference.
[0073] In certain embodiments the D2SP is operably linked to a
nucleotide sequence encoding a recombinase. Any suitable
recombinase that may be operably linked to the D2SP can be used.
Suitable recombinases include, but are not limited to Cre and Flp
recombinases, and derivatives thereof. The recombinases and use
thereof in inducing site-specific recombination with a target
nucleic acid are described, e.g., in US App. Pub. Nos. 20130019325
and 20060003443, U.S. Pat. No. 8,518,392 and Wu et al. PLoS One
2009 4:e8054, which are incorporated herein by reference.
Light-Responsive Polypeptides
[0074] As summarized above, aspects of the present disclosure
include a D2SP operably linked to a nucleotide sequence encoding a
light-responsive polypeptide. The light-activated ion channel
polypeptides are adapted to allow one or more ions to pass through
the plasma membrane of a target cell when the polypeptide is
illuminated with light of an activating wavelength. Light-activated
proteins may be characterized as ion pump proteins, which
facilitate the passage of a small number of ions through the plasma
membrane per photon of light, or as ion channel proteins, which
allow a stream of ions to freely flow through the plasma membrane
when the channel is open. In some embodiments, the light-responsive
polypeptide depolarizes the target cell when activated by light of
an activating wavelength. In some embodiments, the light-responsive
polypeptide hyperpolarizes the target cell when activated by light
of an activating wavelength.
[0075] In some embodiments, the light-responsive polypeptides are
activated by blue light. In some embodiments, the light-responsive
polypeptides are activated by green light. In some embodiments, the
light-responsive polypeptides are activated by yellow light. In
some embodiments, the light-responsive polypeptides are activated
by orange light. In some embodiments, the light-responsive
polypeptides are activated by red light.
[0076] In some embodiments, the light-responsive polypeptide
expressed in a cell can be fused to one or more amino acid sequence
motifs selected from the group consisting of a signal peptide, an
endoplasmic reticulum (ER) export signal, a membrane trafficking
signal, and/or an N-terminal golgi export signal. The one or more
amino acid sequence motifs which enhance light-responsive protein
transport to the plasma membranes of mammalian cells can be fused
to the N-terminus, the C-terminus, or to both the N- and C-terminal
ends of the light-responsive polypeptide. In some cases, the one or
more amino acid sequence motifs which enhance light-responsive
polypeptide transport to the plasma membranes of mammalian cells is
fused internally within a light-responsive polypeptide. Optionally,
the light-responsive polypeptide and the one or more amino acid
sequence motifs may be separated by a linker.
[0077] In some embodiments, the light-responsive polypeptide can be
modified by the addition of a trafficking signal (ts) which
enhances transport of the protein to the cell plasma membrane. In
some embodiments, the trafficking signal can be derived from the
amino acid sequence of the human inward rectifier potassium channel
Kir2.1. In other embodiments, the trafficking signal can comprise
the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56).
Trafficking sequences that are suitable for use can comprise an
amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity to
an amino acid sequence such a trafficking sequence of human inward
rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ
ID NO: 56)).
[0078] A trafficking sequence can have a length of from about 10
amino acids to about 50 amino acids, e.g., from about 10 amino
acids to about 20 amino acids, from about 20 amino acids to about
30 amino acids, from about 30 amino acids to about 40 amino acids,
or from about 40 amino acids to about 50 amino acids.
[0079] ER export sequences that are suitable for use with a
light-responsive polypeptide include, e.g., VXXSL (where X is any
amino acid) (e.g., VKESL (SEQ ID NO: 57); VLGSL (SEQ ID NO: 58);
etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE (SEQ ID NO: 60)
(where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 61); and the
like. An ER export sequence can have a length of from about 5 amino
acids to about 25 amino acids, e.g., from about 5 amino acids to
about 10 amino acids, from about 10 amino acids to about 15 amino
acids, from about 15 amino acids to about 20 amino acids, or from
about 20 amino acids to about 25 amino acids.
[0080] Signal sequences that are suitable for use can comprise an
amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity to
an amino acid sequence such as one of the following: 1) the signal
peptide of hChR2 (e.g., MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO:
62)); 2) the 32 subunit signal peptide of the neuronal nicotinic
acetylcholine receptor (e.g., MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID
NO: 63)); 3) a nicotinic acetylcholine receptor signal sequence
(e.g., MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO: 64)); and 4) a nicotinic
acetylcholine receptor signal sequence (e.g., MRGTPLLLVVSLFSLLQD
(SEQ ID NO: 65)).
[0081] A signal sequence can have a length of from about 10 amino
acids to about 50 amino acids, e.g., from about 10 amino acids to
about 20 amino acids, from about 20 amino acids to about 30 amino
acids, from about 30 amino acids to about 40 amino acids, or from
about 40 amino acids to about 50 amino acids.
[0082] In some embodiments, the signal peptide sequence in the
protein can be deleted or substituted with a signal peptide
sequence from a different protein.
[0083] Exemplary light-responsive polypeptides are described in,
e.g., PCT App. No. PCT/US2011/028893, which is incorporated herein
by reference. Representative light-responsive polypeptides that
find use in the present disclosure are further described below.
Depolarizing Light-Responsive Polypeptides
ChR
[0084] In some aspects, a depolarizing light-responsive polypeptide
is derived from Chlamydomonas reinhardtii, wherein the polypeptide
is capable of transporting cations across a cell membrane when the
cell is illuminated with light. In another embodiment, the
light-responsive polypeptide comprise an amino acid sequence at
least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% identical to the sequence shown in SEQ ID NO: 4. The light
used to activate the light-responsive cation channel protein
derived from Chlamydomonas reinhardtii can have a wavelength
between about 460 and about 495 nm or can have a wavelength of
about 480 nm. Additionally, light pulses having a temporal
frequency of about 100 Hz can be used to activate the
light-responsive protein. In some embodiments, activation of the
light-responsive cation channel derived from Chlamydomonas
reinhardtii with light pulses having a temporal frequency of about
100 Hz can cause depolarization of the neurons expressing the
light-responsive cation channel. The light-responsive cation
channel protein can additionally comprise substitutions, deletions,
and/or insertions introduced into a native amino acid sequence to
increase or decrease sensitivity to light, increase or decrease
sensitivity to particular wavelengths of light, and/or increase or
decrease the ability of the light-responsive cation channel protein
to regulate the polarization state of the plasma membrane of the
cell. Additionally, the light-responsive cation channel protein can
comprise one or more conservative amino acid substitutions and/or
one or more non-conservative amino acid substitutions. The
light-responsive proton pump protein containing substitutions,
deletions, and/or insertions introduced into the native amino acid
sequence suitably retains the ability to transport cations across a
cell membrane.
[0085] In some embodiments, the light-responsive cation channel
includes a T159C substitution of the amino acid sequence set forth
in SEQ ID NO: 4. In some embodiments, the light-responsive cation
channel includes a L132C substitution of the amino acid sequence
set forth in SEQ ID NO: 4. In some embodiments, the
light-responsive cation channel includes an E123T substitution of
the amino acid sequence set forth in SEQ ID NO: 4. In some
embodiments, the light-responsive cation channel includes an E123A
substitution of the amino acid sequence set forth in SEQ ID NO: 4.
In some embodiments, the light-responsive cation channel includes a
T159C substitution and an E123T substitution of the amino acid
sequence set forth in SEQ ID NO: 4. In some embodiments, the
light-responsive cation channel includes a T159C substitution and
an E123A substitution of the amino acid sequence set forth in SEQ
ID NO: 4. In some embodiments, the light-responsive cation channel
includes a T159C substitution, an L132C substitution, and an E123T
substitution of the amino acid sequence set forth in SEQ ID NO: 4.
In some embodiments, the light-responsive cation channel includes a
T159C substitution, an L132C substitution, and an E123A
substitution of the amino acid sequence set forth in SEQ ID NO: 4.
In some embodiments, the light-responsive cation channel includes
an L132C substitution and an E123T substitution of the amino acid
sequence set forth in SEQ ID NO: 4. In some embodiments, the
light-responsive cation channel includes an L132C substitution and
an E123A substitution of the amino acid sequence set forth in SEQ
ID NO: 4.
[0086] In some embodiments, a ChR2 protein comprises at least one
(such as one, two, three, or more) amino acid sequence motifs that
enhance transport to the plasma membranes of target cells selected
from the group consisting of a signal peptide, an ER export signal,
and a membrane trafficking signal. In some embodiments, the ChR2
protein comprises an N-terminal signal peptide and a C-terminal ER
export signal. In some embodiments, the ChR2 protein comprises an
N-terminal signal peptide and a C-terminal trafficking signal. In
some embodiments, the ChR2 protein comprises an N-terminal signal
peptide, a C-terminal ER export signal, and a C-terminal
trafficking signal. In some embodiments, the ChR2 protein comprises
a C-terminal ER export signal and a C-terminal trafficking signal.
In some embodiments, the C-terminal ER export signal and the
C-terminal trafficking signal are linked by a linker. The linker
can comprise any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150,
175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length.
The linker may further comprise a fluorescent protein, for example,
but not limited to, a yellow fluorescent protein, a red fluorescent
protein, a green fluorescent protein, or a cyan fluorescent
protein. In some embodiments the ER export signal is more
C-terminally located than the trafficking signal. In some
embodiments the trafficking signal is more C-terminally located
than the ER Export signal.
[0087] In some embodiments, the trafficking signal can be derived
from the amino acid sequence of the human inward rectifier
potassium channel Kir2.1. In other embodiments, the trafficking
signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV
(SEQ ID NO: 56). Trafficking sequences that are suitable for use
can comprise an amino acid sequence having at least 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid
sequence identity to an amino acid sequence such a trafficking
sequence of human inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0088] In certain embodiments, the ChR2 protein can have an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 5.
[0089] In other embodiments, the light-responsive polypeptide is a
step function opsin (SFO) protein or a stabilized step function
opsin (SSFO) protein that can have specific amino acid
substitutions at key positions in the retinal binding pocket of the
protein. In some embodiments, the SFO protein can have a mutation
at amino acid residue C128 of SEQ ID NO: 4. In other embodiments,
the SFO protein has a C128A mutation in SEQ ID NO: 4. In other
embodiments, the SFO protein has a C128S mutation in SEQ ID NO: 4.
In another embodiment, the SFO protein has a C128T mutation in SEQ
ID NO: 4.
[0090] In some embodiments, the SSFO protein can have a mutation at
amino acid residue D156 of SEQ ID NO: 4. In other embodiments, the
SSFO protein can have a mutation at both amino acid residues C128
and D156 of SEQ ID NO: 4. In one embodiment, the SSFO protein has
an C128S and a D156A mutation in SEQ ID NO: 4. In another
embodiment, the SSFO protein can comprise an amino acid sequence at
least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% identical to the sequence shown in SEQ ID NO: 6; and
includes an alanine, serine, or threonine at amino acid 128; and
includes a alanine at amino acid 156. In another embodiment, the
SSFO protein can comprise a C128T mutation in SEQ ID NO: 4. In some
embodiments, the SSFO protein includes C128T and D156A mutations in
SEQ ID NO: 6.
[0091] In some embodiments the SFO or SSFO proteins provided herein
can be capable of mediating a depolarizing current in the cell when
the cell is illuminated with blue light. In other embodiments, the
light can have a wavelength of about 445 nm. Additionally, in some
embodiments the light can be delivered as a single pulse of light
or as spaced pulses of light due to the prolonged stability of SFO
and SSFO photocurrents. In some embodiments, activation of the SFO
or SSFO protein with single pulses or spaced pulses of light can
cause depolarization of a neuron expressing the SFO or SSFO
protein. In some embodiments, each of the disclosed step function
opsin and stabilized step function opsin proteins can have specific
properties and characteristics for use in depolarizing the membrane
of a neuronal cell in response to light.
[0092] Further disclosure related to SFO or SSFO proteins can be
found in International Patent Application Publication No. WO
2010/056970, the disclosure of which is hereby incorporated by
reference in its entirety.
[0093] In some cases, the ChR2-based SFO or SSFO comprises a
membrane trafficking signal and/or an ER export signal. In some
embodiments, the trafficking signal is derived from the amino acid
sequence of the human inward rectifier potassium channel Kir2.1. In
other embodiments, the trafficking signal comprises the amino acid
sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Trafficking
sequences that are suitable for use comprises an amino acid
sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%, amino acid sequence identity to an amino
acid sequence such a trafficking sequence of human inward rectifier
potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In some cases, the ER export signal is, e.g., VXXSL (where X
is any amino acid) (e.g., VKESL (SEQ ID NO: 57), VLGSL (SEQ ID NO:
58); etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE (SEQ ID NO:
60) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 61); and
the like.
[0094] In certain embodiments, the SSFO protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 7.
Volvox carteri Light-Responsive Polypeptide
[0095] In some embodiments, a suitable light-responsive polypeptide
is a cation channel derived from Volvox carteri (VChR1) and is
activated by illumination with light of a wavelength of from about
500 nm to about 600 nm, e.g., from about 525 nm to about 550 nm,
e.g., 545 nm. In some embodiments, the light-responsive ion channel
protein comprises an amino acid sequence at least 75%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
the sequence shown in SEQ ID NO: 8. The light-responsive ion
channel protein can additionally comprise substitutions, deletions,
and/or insertions introduced into a native amino acid sequence to
increase or decrease sensitivity to light, increase or decrease
sensitivity to particular wavelengths of light, and/or increase or
decrease the ability of the light-responsive ion channel protein to
regulate the polarization state of the plasma membrane of the cell.
Additionally, the light-responsive ion channel protein can comprise
one or more conservative amino acid substitutions and/or one or
more non-conservative amino acid substitutions. The
light-responsive ion channel protein containing substitutions,
deletions, and/or insertions introduced into the native amino acid
sequence suitably retains the ability to transport ions across the
plasma membrane of a neuronal cell in response to light.
[0096] In some cases, a VChR1 light-responsive cation channel
protein comprises a core amino acid sequence at least 75%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical
to the sequence shown in SEQ ID NO: 8 and at least one (such as
one, two, three, or more) amino acid sequence motifs which enhance
transport to the plasma membranes of mammalian cells selected from
the group consisting of a signal peptide, an ER export signal, and
a membrane trafficking signal. In some embodiments, the
light-responsive proton ion channel comprises an N-terminal signal
peptide and a C-terminal ER export signal. In some embodiments, the
light-responsive ion channel protein comprises an N-terminal signal
peptide and a C-terminal trafficking signal. In some embodiments,
the light-responsive ion channel protein comprises an N-terminal
signal peptide, a C-terminal ER Export signal, and a C-terminal
trafficking signal. In some embodiments, the light-responsive ion
channel protein comprises a C-terminal ER Export signal and a
C-terminal trafficking signal. In some embodiments, the C-terminal
ER Export signal and the C-terminal trafficking signal are linked
by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50,
75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. The linker may further comprise a fluorescent
protein, for example, but not limited to, a yellow fluorescent
protein, a red fluorescent protein, a green fluorescent protein, or
a cyan fluorescent protein. In some embodiments the ER Export
signal is more C-terminally located than the trafficking signal. In
some embodiments the trafficking signal is more C-terminally
located than the ER Export signal.
[0097] In some embodiments, the trafficking signal is derived from
the amino acid sequence of the human inward rectifier potassium
channel Kir2.1. In other embodiments, the trafficking signal
comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56). Trafficking sequences that are suitable for use can comprise
an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity
to an amino acid sequence such a trafficking sequence of human
inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0098] In certain embodiments, the VChR1protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 9.
Step Function Opsins and Stabilized Step Function Opsins Based on
VChR1
[0099] In other embodiments, the light-responsive polypeptide is a
SFO or an SSFO based on VChR1. In some embodiments, the SFO protein
can have a mutation at amino acid residue C123 of SEQ ID NO: 8. In
other embodiments, the SFO protein has a C123A mutation in SEQ ID
NO: 8. In other embodiments, the SFO protein has a C123S mutation
in SEQ ID NO: 8. In another embodiment, the SFO protein has a C123T
mutation in SEQ ID NO: 8.
[0100] In some embodiments, the SFO protein can have a mutation at
amino acid residue D151 of SEQ ID NO: 8. In other embodiments, the
SFO protein can have a mutation at both amino acid residues C123
and D151 of SEQ ID NO: 8. In one embodiment, the SFO protein has an
C123S and a D151A mutation in SEQ ID NO: 8.
[0101] In some embodiments an SFO or SSFO protein is capable of
mediating a depolarizing current in the cell when the cell is
illuminated with blue light. In some embodiments, the light has a
wavelength of about 560 nm. Additionally, in some embodiments the
light is delivered as a single pulse of light or as spaced pulses
of light due to the prolonged stability of SFO and SSFO
photocurrents. In some embodiments, activation of the SFO or SSFO
protein with single pulses or spaced pulses of light can cause
depolarization of a neuron expressing the SFO or SSFO protein. In
some embodiments, each of the disclosed step function opsin and
stabilized step function opsin proteins can have specific
properties and characteristics for use in depolarizing the membrane
of a neuronal cell in response to light.
[0102] In some cases, the VChR1-based SFO or SSFO comprises a
membrane trafficking signal and/or an ER export signal. In some
embodiments, the trafficking signal can be derived from the amino
acid sequence of the human inward rectifier potassium channel
Kir2.1. Trafficking sequences that are suitable for use can
comprise an amino acid sequence having at least 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence
identity to an amino acid sequence such a trafficking sequence of
human inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
C1V1 Chimeric Cation Channels
[0103] In other embodiments, the light-responsive cation channel
protein is a C1V1 chimeric protein derived from the VChR1 protein
of Volvox carteri and the ChR1 protein from Chlamydomonas
reinhardti, wherein the protein comprises the amino acid sequence
of VChR1 having at least the first and second transmembrane helices
replaced by the first and second transmembrane helices of ChR1; is
responsive to light; and is capable of mediating a depolarizing
current in the cell when the cell is illuminated with light. In
some embodiments, the C1V1 protein further comprises a replacement
within the intracellular loop domain located between the second and
third transmembrane helices of the chimeric light responsive
protein, wherein at least a portion of the intracellular loop
domain is replaced by the corresponding portion from ChR1. In
another embodiment, the portion of the intracellular loop domain of
the C1V1 chimeric protein can be replaced with the corresponding
portion from ChR1 extending to amino acid residue A145 of the ChR1.
In other embodiments, the C1V1 chimeric protein further comprises a
replacement within the third transmembrane helix of the chimeric
light responsive protein, wherein at least a portion of the third
transmembrane helix is replaced by the corresponding sequence of
ChR1. In yet another embodiment, the portion of the intracellular
loop domain of the C1V1 chimeric protein can be replaced with the
corresponding portion from ChR1 extending to amino acid residue
W163 of the ChR1. In other embodiments, the C1V1 chimeric protein
comprises an amino acid sequence at least 75%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
sequence shown in SEQ ID NO: 10.
[0104] In some embodiments, the C1V1 protein mediates a
depolarizing current in the cell when the cell is illuminated with
green light. In some embodiments, the light has a wavelength of
between about 540 nm to about 560 nm. In some embodiments, the
light can have a wavelength of about 542 nm. In some embodiments,
the C1V1 chimeric protein is not capable of mediating a
depolarizing current in the cell when the cell is illuminated with
violet light. In some embodiments, the chimeric protein is not
capable of mediating a depolarizing current in the cell when the
cell is illuminated with light having a wavelength of about 405 nm.
Additionally, in some embodiments, light pulses having a temporal
frequency of about 100 Hz can be used to activate the C1V1
protein.
[0105] In some cases, the C1V1 polypeptide comprises a membrane
trafficking signal and/or an ER export signal. In some embodiments,
the trafficking signal is derived from the amino acid sequence of
the human inward rectifier potassium channel Kir2.1. In other
embodiments, the trafficking signal comprises the amino acid
sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Trafficking
sequences that are suitable for use can comprise an amino acid
sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%, amino acid sequence identity to an amino
acid sequence such a trafficking sequence of human inward rectifier
potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In some cases, the ER export signal is, e.g., VXXSL (where X
is any amino acid) (e.g., VKESL (SEQ ID NO: 57), VLGSL (SEQ ID NO:
58); etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE (SEQ ID NO:
60) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 61); and
the like.
[0106] In certain embodiments, the C1V1 protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 11.
C1V1 Variants
[0107] In some aspects, a suitable light-responsive polypeptide
comprises substituted or mutated amino acid sequences, wherein the
mutant polypeptide retains the characteristic light-activatable
nature of the precursor C1V1 chimeric polypeptide but may also
possess altered properties in some specific aspects. For example,
the mutant light-responsive C1V1 chimeric proteins described herein
can exhibit an increased level of expression both within an animal
cell or on the animal cell plasma membrane; an altered
responsiveness when exposed to different wavelengths of light,
particularly red light; and/or a combination of traits whereby the
chimeric C1V1 polypeptide possess the properties of low
desensitization, fast deactivation, low violet-light activation for
minimal cross-activation with other light-responsive cation
channels, and/or strong expression in animal cells.
[0108] Accordingly, suitable light-responsive proteins include C1V1
chimeric light-responsive proteins that can have specific amino
acid substitutions at key positions throughout the retinal binding
pocket of the VChR1 portion of the chimeric polypeptide. In some
embodiments, the C1V1 protein comprises an amino acid substitution
at amino acid residue E122 of SEQ ID NO: 10. In some embodiments,
the C1V1 protein comprises a substitution at amino acid residue
E162 of SEQ ID NO: 10. In other embodiments, the C1V1 protein
comprises a substitution at both amino acid residues E162 and E122
of SEQ ID NO: 10.
[0109] In some aspects, the C1V1-E122 mutant chimeric protein is
capable of mediating a depolarizing current in the cell when the
cell is illuminated with light. In some embodiments the light is
green light. In other embodiments, the light has a wavelength of
between about 540 nm to about 560 nm. In some embodiments, the
light has a wavelength of about 546 nm. In other embodiments, the
C1V1-E122 mutant chimeric protein mediates a depolarizing current
in the cell when the cell is illuminated with red light. In some
embodiments, the red light has a wavelength of about 630 nm. In
some embodiments, the C1V1-E122 mutant chimeric protein does not
mediate a depolarizing current in the cell when the cell is
illuminated with violet light. In some embodiments, the chimeric
protein does not mediate a depolarizing current in the cell when
the cell is illuminated with light having a wavelength of about 405
nm. Additionally, in some embodiments, light pulses having a
temporal frequency of about 100 Hz can be used to activate the
C1V1-E122 mutant chimeric protein. In some embodiments, activation
of the C1V1-E122 mutant chimeric protein with light pulses having a
frequency of 100 Hz can cause depolarization of the neurons
expressing the C1V1-E122 mutant chimeric protein.
[0110] In other aspects, the C1V1-E162 mutant chimeric protein is
capable of mediating a depolarizing current in the cell when the
cell is illuminated with light. In some embodiments the light can
be green light. In other embodiments, the light can have a
wavelength of between about 535 nm to about 540 nm. In some
embodiments, the light can have a wavelength of about 542 nm. In
other embodiments, the light can have a wavelength of about 530 nm.
In some embodiments, the C1V1-E162 mutant chimeric protein does not
mediate a depolarizing current in the cell when the cell is
illuminated with violet light. In some embodiments, the chimeric
protein does not mediate a depolarizing current in the cell when
the cell is illuminated with light having a wavelength of about 405
nm. Additionally, in some embodiments, light pulses having a
temporal frequency of about 100 Hz can be used to activate the
C1V1-E162 mutant chimeric protein. In some embodiments, activation
of the C1V1-E162 mutant chimeric protein with light pulses having a
frequency of 100 Hz can cause depolarization-induced synaptic
depletion of the neurons expressing the C1V1-E162 mutant chimeric
protein.
[0111] In yet other aspects, the C1V1-E122/E162 mutant chimeric
protein is capable of mediating a depolarizing current in the cell
when the cell is illuminated with light. In some embodiments the
light can be green light. In other embodiments, the light can have
a wavelength of between about 540 nm to about 560 nm. In some
embodiments, the light can have a wavelength of about 546 nm. In
some embodiments, the C1V1-E122/E162 mutant chimeric protein does
not mediate a depolarizing current in the cell when the cell is
illuminated with violet light. In some embodiments, the chimeric
protein does not mediate a depolarizing current in the cell when
the cell is illuminated with light having a wavelength of about 405
nm. In some embodiments, the C1V1-E122/E162 mutant chimeric protein
can exhibit less activation when exposed to violet light relative
to C1V1 chimeric proteins lacking mutations at E122/E162 or
relative to other light-responsive cation channel proteins.
Additionally, in some embodiments, light pulses having a temporal
frequency of about 100 Hz can be used to activate the
C1V1-E122/E162 mutant chimeric protein. In some embodiments,
activation of the C1V1-E122/E162 mutant chimeric protein with light
pulses having a frequency of 100 Hz can cause
depolarization-induced synaptic depletion of the neurons expressing
the C1V1-E122/E162 mutant chimeric protein.
[0112] In some cases, the C1V1 variant polypeptide comprises a
membrane trafficking signal and/or an ER export signal. In some
embodiments, the trafficking signal can be derived from the amino
acid sequence of the human inward rectifier potassium channel
Kir2.1. In other embodiments, the trafficking signal comprises the
amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56).
Trafficking sequences that are suitable for use can comprise an
amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity to
an amino acid sequence such a trafficking sequence of human inward
rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ
ID NO: 56)). In some cases, the ER export signal is, e.g., VXXSL
(where X is any amino acid) (e.g., VKESL (SEQ ID NO: 57), VLGSL
(SEQ ID NO: 58); etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE
(SEQ ID NO: 60) (where X is any amino acid), e.g., FCYENEV (SEQ ID
NO: 61); and the like.
C1C2 Chimeric Cation Channels
[0113] In other embodiments, the light-responsive cation channel
protein is a C1C2 chimeric protein derived from the ChR1 and the
ChR2 proteins from Chlamydomonas reinhardti, wherein the protein is
responsive to light and is capable of mediating a depolarizing
current in the cell when the cell is illuminated with light. In
another embodiment, the light-responsive polypeptide comprises an
amino acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 12. The light-responsive cation channel protein can
additionally comprise substitutions, deletions, and/or insertions
introduced into a native amino acid sequence to increase or
decrease sensitivity to light, increase or decrease sensitivity to
particular wavelengths of light, and/or increase or decrease the
ability of the light-responsive cation channel protein to regulate
the polarization state of the plasma membrane of the cell.
Additionally, the light-responsive cation channel protein comprises
one or more conservative amino acid substitutions and/or one or
more non-conservative amino acid substitutions. The
light-responsive proton pump protein containing substitutions,
deletions, and/or insertions introduced into the native amino acid
sequence suitably retains the ability to transport cations across a
cell membrane.
[0114] In some embodiments, a C1C2 protein comprises at least one
(such as one, two, three, or more) amino acid sequence motifs that
enhance transport to the plasma membranes of target cells selected
from the group consisting of a signal peptide, an ER export signal,
and a membrane trafficking signal. In some embodiments, the C1C2
protein comprises an N-terminal signal peptide and a C-terminal ER
export signal. In some embodiments, the C1C2 protein comprises an
N-terminal signal peptide and a C-terminal trafficking signal. In
some embodiments, the C1C2 protein comprises an N-terminal signal
peptide, a C-terminal ER export signal, and a C-terminal
trafficking signal. In some embodiments, the C1C2 protein comprises
a C-terminal ER export signal and a C-terminal trafficking signal.
In some embodiments, the C-terminal ER export signal and the
C-terminal trafficking signal are linked by a linker. The linker
can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175,
200, 225, 250, 275, 300, 400, or 500 amino acids in length. The
linker may further comprise a fluorescent protein, for example, but
not limited to, a yellow fluorescent protein, a red fluorescent
protein, a green fluorescent protein, or a cyan fluorescent
protein. In some embodiments the ER export signal is more
C-terminally located than the trafficking signal. In some
embodiments the trafficking signal is more C-terminally located
than the ER Export signal.
[0115] In some embodiments, the trafficking signal can be derived
from the amino acid sequence of the human inward rectifier
potassium channel Kir2.1. In other embodiments, the trafficking
signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV
(SEQ ID NO: 56). Trafficking sequences that are suitable for use
can comprise an amino acid sequence having at least 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid
sequence identity to an amino acid sequence such a trafficking
sequence of human inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0116] In certain embodiments, the C1C2 protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 13.
ReaChR
[0117] In some aspects, a depolarizing light-responsive polypeptide
is a red shifted variant of a depolarizing light-responsive
polypeptide derived from Chlamydomonas reinhardtii; such
light-responsive polypeptides are referred to herein as a "ReaChR
polypeptide" or "ReaChR protein" or "ReaChR." In another
embodiment, the light-responsive polypeptide comprises an amino
acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID
NO: 14. The light used to activate the ReaChR polypeptide can have
a wavelength between about 590 and about 630 nm or can have a
wavelength of about 610 nm. The ReaChR protein can additionally
comprise substitutions, deletions, and/or insertions introduced
into a native amino acid sequence to increase or decrease
sensitivity to light, increase or decrease sensitivity to
particular wavelengths of light, and/or increase or decrease the
ability of the light-responsive cation channel protein to regulate
the polarization state of the plasma membrane of the cell.
Additionally, the ReaChR protein can comprise one or more
conservative amino acid substitutions and/or one or more
non-conservative amino acid substitutions. The ReaChR containing
substitutions, deletions, and/or insertions introduced into the
native amino acid sequence suitably retains the ability to
transport cations across a cell membrane.
[0118] In some embodiments, a ReaChR protein comprises at least one
(such as one, two, three, or more) amino acid sequence motifs that
enhance transport to the plasma membranes of target cells selected
from the group consisting of a signal peptide, an ER export signal,
and a membrane trafficking signal. In some embodiments, the ReaChR
protein comprises an N-terminal signal peptide and a C-terminal ER
export signal. In some embodiments, the ReaChR protein comprises an
N-terminal signal peptide and a C-terminal trafficking signal. In
some embodiments, the ReaChR protein comprises an N-terminal signal
peptide, a C-terminal ER export signal, and a C-terminal
trafficking signal. In some embodiments, the ReaChR protein
comprises a C-terminal ER export signal and a C-terminal
trafficking signal. In some embodiments, the C-terminal ER export
signal and the C-terminal trafficking signal are linked by a
linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. The linker may further comprise a fluorescent
protein, for example, but not limited to, a yellow fluorescent
protein, a red fluorescent protein, a green fluorescent protein, or
a cyan fluorescent protein. In some embodiments the ER export
signal is more C-terminally located than the trafficking signal. In
some embodiments the trafficking signal is more C-terminally
located than the ER Export signal.
[0119] In some embodiments, the trafficking signal can be derived
from the amino acid sequence of the human inward rectifier
potassium channel Kir2.1. In other embodiments, the trafficking
signal can comprise the amino acid sequence KSRITSEGEYIPLDQIDINV
(SEQ ID NO: 56). Trafficking sequences that are suitable for use
can comprise an amino acid sequence having at least 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid
sequence identity to an amino acid sequence such a trafficking
sequence of human inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0120] In certain embodiments, the ReaChR protein comprises an
amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence
shown in SEQ ID NO: 15.
SdChR
[0121] In some aspects, a depolarizing light-responsive polypeptide
is a SdChR polypeptide derived from Scherffelia dubia, wherein the
SdChR polypeptide is capable of transporting cations across a cell
membrane when the cell is illuminated with light. In some cases,
the SdChR polypeptide comprises an amino acid sequence at least
75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the sequence shown in SEQ ID NO: 16. The light used to
activate the SdChR polypeptide can have a wavelength between about
440 and about 490 nm or can have a wavelength of about 460 nm. The
SdChR protein can additionally comprise substitutions, deletions,
and/or insertions introduced into a native amino acid sequence to
increase or decrease sensitivity to light, increase or decrease
sensitivity to particular wavelengths of light, and/or increase or
decrease the ability of the SdChR protein to regulate the
polarization state of the plasma membrane of the cell. In some
instances, the SdChR protein comprises one or more conservative
amino acid substitutions and/or one or more non-conservative amino
acid substitutions. The SdChR protein containing substitutions,
deletions, and/or insertions introduced into the native amino acid
sequence suitably retains the ability to transport cations across a
cell membrane.
[0122] In some embodiments, a SdChR protein comprises at least one
(such as one, two, three, or more) amino acid sequence motifs that
enhance transport to the plasma membranes of target cells selected
from the group consisting of a signal peptide, an ER export signal,
and a membrane trafficking signal. In some embodiments, the SdChR
protein comprises an N-terminal signal peptide and a C-terminal ER
export signal. In some embodiments, the SdChR protein comprises an
N-terminal signal peptide and a C-terminal trafficking signal. In
some embodiments, the SdChR protein comprises an N-terminal signal
peptide, a C-terminal ER export signal, and a C-terminal
trafficking signal. In some embodiments, the SdChR protein
comprises a C-terminal ER export signal and a C-terminal
trafficking signal. In some embodiments, the C-terminal ER export
signal and the C-terminal trafficking signal are linked by a
linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. The linker may further comprise a fluorescent
protein, for example, but not limited to, a yellow fluorescent
protein, a red fluorescent protein, a green fluorescent protein, or
a cyan fluorescent protein. In some embodiments the ER export
signal is more C-terminally located than the trafficking signal. In
some embodiments the trafficking signal is more C-terminally
located than the ER Export signal.
[0123] In some embodiments, the trafficking signal can be derived
from the amino acid sequence of the human inward rectifier
potassium channel Kir2.1. In other embodiments, the trafficking
signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ
ID NO: 56). Trafficking sequences that are suitable for use
comprises an amino acid sequence having at least 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid
sequence identity to an amino acid sequence such a trafficking
sequence of human inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0124] In certain embodiments, the SdChR protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 17.
CnChR1
[0125] In some aspects, a depolarizing light-responsive polypeptide
can be, e.g. CnChR1, derived from Chlamydomonas noctigama, wherein
the CnChR1 polypeptide is capable of transporting cations across a
cell membrane when the cell is illuminated with light. In some
cases, the CnChR1 polypeptide comprises an amino acid sequence at
least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% identical to the sequence shown in SEQ ID NO: 18. The light
used to activate the CnChR1 polypeptide can have a wavelength
between about 560 and about 630 nm or can have a wavelength of
about 600 nm. The CnChR1 protein can additionally comprise
substitutions, deletions, and/or insertions introduced into a
native amino acid sequence to increase or decrease sensitivity to
light, increase or decrease sensitivity to particular wavelengths
of light, and/or increase or decrease the ability of the CnChR1
protein to regulate the polarization state of the plasma membrane
of the cell. In some cases, the CnChR1 protein comprises one or
more conservative amino acid substitutions and/or one or more
non-conservative amino acid substitutions. The CnChR1 protein
containing substitutions, deletions, and/or insertions introduced
into the native amino acid sequence suitably retains the ability to
transport cations across a cell membrane.
[0126] In some embodiments, a CnChR1protein comprises at least one
(such as one, two, three, or more) amino acid sequence motifs that
enhance transport to the plasma membranes of target cells selected
from the group consisting of a signal peptide, an ER export signal,
and a membrane trafficking signal. In some embodiments, the
CnChR1protein includes an N-terminal signal peptide and a
C-terminal ER export signal. In some embodiments, the CnChR1protein
includes an N-terminal signal peptide and a C-terminal trafficking
signal. In some embodiments, the CnChR1protein comprises an
N-terminal signal peptide, a C-terminal ER export signal, and a
C-terminal trafficking signal. In some embodiments, the
CnChR1protein comprises a C-terminal ER export signal and a
C-terminal trafficking signal. In some embodiments, the C-terminal
ER export signal and the C-terminal trafficking signal are linked
by a linker. The linker can be any of about 5, 10, 20, 30, 40, 50,
75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. The linker may further comprise a fluorescent
protein, for example, but not limited to, a yellow fluorescent
protein, a red fluorescent protein, a green fluorescent protein, or
a cyan fluorescent protein. In some embodiments the ER export
signal is more C-terminally located than the trafficking signal. In
some embodiments the trafficking signal is more C-terminally
located than the ER Export signal.
[0127] In some embodiments, the trafficking signal is derived from
the amino acid sequence of the human inward rectifier potassium
channel Kir2.1. In other embodiments, the trafficking signal
comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56). Trafficking sequences that are suitable for use can comprise
an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity
to an amino acid sequence such a trafficking sequence of human
inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0128] In certain embodiments, the CnChR1protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 19.
CsChrimson
[0129] In other embodiments, the light-responsive cation channel
protein is a CsChrimson chimeric protein derived from a CsChR
protein of Chloromonas subdivisa and CnChR1 protein from
Chlamydomonas noctigama, wherein the N terminus of the protein
comprises the amino acid sequence of residues 1-73 of CsChR
followed by residues 79-350 of the amino acid sequence of CnChR1;
is responsive to light; and is capable of mediating a depolarizing
current in the cell when the cell is illuminated with light. In
another embodiment, the CsChrimson polypeptide comprises an amino
acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID
NO: 20. The CsChrimson protein can additionally comprise
substitutions, deletions, and/or insertions introduced into a
native amino acid sequence to increase or decrease sensitivity to
light, increase or decrease sensitivity to particular wavelengths
of light, and/or increase or decrease the ability of the CsChrimson
protein to regulate the polarization state of the plasma membrane
of the cell. Additionally, the CsChrimson protein can comprise one
or more conservative amino acid substitutions and/or one or more
non-conservative amino acid substitutions. A CsChrimson protein
containing substitutions, deletions, and/or insertions introduced
into the native amino acid sequence suitably retains the ability to
transport cations across a cell membrane.
[0130] In some embodiments, a CsChrimson protein comprises at least
one (such as one, two, three, or more) amino acid sequence motifs
that enhance transport to the plasma membranes of target cells
selected from the group consisting of a signal peptide, an ER
export signal, and a membrane trafficking signal. In some
embodiments, the CsChrimson protein comprises an N-terminal signal
peptide and a C-terminal ER export signal. In some embodiments, the
CsChrimson protein comprises an N-terminal signal peptide and a
C-terminal trafficking signal. In some embodiments, the CsChrimson
protein comprises an N-terminal signal peptide, a C-terminal ER
export signal, and a C-terminal trafficking signal. In some
embodiments, the CsChrimson protein comprises a C-terminal ER
export signal and a C-terminal trafficking signal. In some
embodiments, the C-terminal ER export signal and the C-terminal
trafficking signal are linked by a linker. The linker can be any of
about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,
275, 300, 400, or 500 amino acids in length. The linker may further
comprise a fluorescent protein, for example, but not limited to, a
yellow fluorescent protein, a red fluorescent protein, a green
fluorescent protein, or a cyan fluorescent protein. In some
embodiments the ER export signal is more C-terminally located than
the trafficking signal. In some embodiments the trafficking signal
is more C-terminally located than the ER Export signal.
[0131] In some embodiments, the trafficking signal is derived from
the amino acid sequence of the human inward rectifier potassium
channel Kir2.1. In other embodiments, the trafficking signal
comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56). Trafficking sequences that are suitable for use can comprise
an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity
to an amino acid sequence such a trafficking sequence of human
inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0132] In certain embodiments, the CsChrimson protein comprises an
amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence
shown in SEQ ID NO: 21.
ShChR1
[0133] In some aspects, a depolarizing light-responsive polypeptide
can be, e.g. ShChR1, derived from Stigeoclonium helveticum, wherein
the ShChR1 polypeptide is capable of transporting cations across a
cell membrane when the cell is illuminated with light. In some
cases, the ShChR1 polypeptide comprises an amino acid sequence at
least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% identical to the sequence shown in SEQ ID NO: 22. The light
used to activate the ShChR1 protein derived from Stigeoclonium
helveticum can have a wavelength between about 480 and about 510 nm
or can have a wavelength of about 500 nm. The ShChR1 protein can
additionally comprise substitutions, deletions, and/or insertions
introduced into a native amino acid sequence to increase or
decrease sensitivity to light, increase or decrease sensitivity to
particular wavelengths of light, and/or increase or decrease the
ability of the ShChR1 protein to regulate the polarization state of
the plasma membrane of the cell. Additionally, the ShChR1 protein
can comprise one or more conservative amino acid substitutions
and/or one or more non-conservative amino acid substitutions. A
ShChR1 protein containing substitutions, deletions, and/or
insertions introduced into the native amino acid sequence suitably
retains the ability to transport cations across a cell
membrane.
[0134] In some embodiments, a ShChR1 protein comprises at least one
(such as one, two, three, or more) amino acid sequence motifs that
enhance transport to the plasma membranes of target cells selected
from the group consisting of a signal peptide, an ER export signal,
and a membrane trafficking signal. In some embodiments, the ShChR1
protein comprises an N-terminal signal peptide and a C-terminal ER
export signal. In some embodiments, the ShChR1 protein comprises an
N-terminal signal peptide and a C-terminal trafficking signal. In
some embodiments, the ShChR1 protein comprises an N-terminal signal
peptide, a C-terminal ER export signal, and a C-terminal
trafficking signal. In some embodiments, the ShChR1protein
comprises a C-terminal ER export signal and a C-terminal
trafficking signal. In some embodiments, the C-terminal ER export
signal and the C-terminal trafficking signal are linked by a
linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. The linker may further comprise a fluorescent
protein, for example, but not limited to, a yellow fluorescent
protein, a red fluorescent protein, a green fluorescent protein, or
a cyan fluorescent protein. In some embodiments the ER export
signal is more C-terminally located than the trafficking signal. In
some embodiments the trafficking signal is more C-terminally
located than the ER Export signal.
[0135] In some embodiments, the trafficking signal can be derived
from the amino acid sequence of the human inward rectifier
potassium channel Kir2.1. In other embodiments, the trafficking
signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ
ID NO: 56). Trafficking sequences that are suitable for use can
comprise an amino acid sequence having at least 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence
identity to an amino acid sequence such a trafficking sequence of
human inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0136] In certain embodiments, the ShChR1 protein comprises an
amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence
shown in SEQ ID NO: 23.
[0137] Other suitable depolarizing light-responsive polypeptides
are described in, e.g., Klapoetke et al. Nat Methods 2014
11:338.
Hyperpolarizing Light-Responsive Polypeptides
Arch
[0138] In some embodiments, a suitable light-responsive polypeptide
is an Archaerhodopsin (Arch) proton pump (e.g., a proton pump
derived from Halorubrum sodomense) that can transport one or more
protons across the plasma membrane of a cell when the cell is
illuminated with light. The light can have a wavelength between
about 530 and about 595 nm or can have a wavelength of about 560
nm. In some embodiments, the Arch protein comprises an amino acid
sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO: 24. The Arch protein can additionally have substitutions,
deletions, and/or insertions introduced into a native amino acid
sequence to increase or decrease sensitivity to light, increase or
decrease sensitivity to particular wavelengths of light, and/or
increase or decrease the ability of the Arch protein to transport
ions across the plasma membrane of a target cell. Additionally, the
Arch protein can comprise one or more conservative amino acid
substitutions and/or one or more non-conservative amino acid
substitutions. An Arch protein containing substitutions, deletions,
and/or insertions introduced into the native amino acid sequence
suitably retains the ability to transport ions across the plasma
membrane of a target cell in response to light.
[0139] In some embodiments, the Arch protein comprises at least one
(such as one, two, three, or more) amino acid sequence motifs
selected from a signal peptide, an ER export signal, and a membrane
trafficking signal, that enhance transport to the plasma membranes
of target cells. In some embodiments, the Arch protein comprises an
N-terminal signal peptide and a C-terminal ER export signal. In
some embodiments, the Arch protein comprises an N-terminal signal
peptide and a C-terminal trafficking signal. In some embodiments,
the Arch protein comprises an N-terminal signal peptide, a
C-terminal ER export signal, and a C-terminal trafficking signal.
In some embodiments, the Arch protein includes a C-terminal ER
export signal and a C-terminal trafficking signal. In some
embodiments, the C-terminal ER export signal and the C-terminal
trafficking signal are linked by a linker. The linker can be any of
about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,
275, 300, 400, or 500 amino acids in length. The linker may further
include a fluorescent protein, for example, but not limited to, a
yellow fluorescent protein, a red fluorescent protein, a green
fluorescent protein, or a cyan fluorescent protein. In some
embodiments the ER export signal is more C-terminally located than
the trafficking signal. In some embodiments the trafficking signal
is more C-terminally located than the ER Export signal.
[0140] In some embodiments, the trafficking signal is derived from
the amino acid sequence of the human inward rectifier potassium
channel Kir2.1. In other embodiments, the trafficking signal can
include the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56). Trafficking sequences that are suitable for use can include an
amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity to
an amino acid sequence such a trafficking sequence of human inward
rectifier potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ
ID NO: 56)). In some cases, the ER export signal is, e.g., VXXSL
(where X is any amino acid) (e.g., VKESL (SEQ ID NO: 57), VLGSL
(SEQ ID NO: 58); etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE
(SEQ ID NO: 60) (where X is any amino acid), e.g., FCYENEV (SEQ ID
NO: 61); and the like.
[0141] In certain embodiments, the Arch protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 25.
ArchT
[0142] In some embodiments, a suitable light-activated protein is
an Archaerhodopsin (ArchT) proton pump (e.g., a proton pump derived
from Halorubrum sp. TP009) that can transport one or more protons
across the plasma membrane of a cell when the cell is illuminated
with light. The light can have a wavelength between about 530 and
about 595 nm or can have a wavelength of about 560 nm. In some
embodiments, the ArchT protein comprises an amino acid sequence
that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 26
(ArchT). The ArchT protein can additionally comprise substitutions,
deletions, and/or insertions introduced into a native amino acid
sequence to increase or decrease sensitivity to light, increase or
decrease sensitivity to particular wavelengths of light, and/or
increase or decrease the ability of the ArchT protein to transport
ions across the plasma membrane of a target cell. Additionally, the
ArchT protein can comprise one or more conservative amino acid
substitutions and/or one or more non-conservative amino acid
substitutions. The ArchT protein containing substitutions,
deletions, and/or insertions introduced into the native amino acid
sequence suitably retains the ability to transport ions across the
plasma membrane of a target cell in response to light.
[0143] In some cases, the ArchT polypeptide comprises a membrane
trafficking signal and/or an ER export signal. In some embodiments,
the trafficking signal can be derived from the amino acid sequence
of the human inward rectifier potassium channel Kir2.1. In other
embodiments, the trafficking signal comprises the amino acid
sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Trafficking
sequences that are suitable for use can comprise an amino acid
sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%, amino acid sequence identity to an amino
acid sequence such a trafficking sequence of human inward rectifier
potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In some cases, the ER export signal is, e.g., VXXSL (where X
is any amino acid) (e.g., VKESL (SEQ ID NO: 57), VLGSL (SEQ ID NO:
58); etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE (SEQ ID NO:
60) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 61); and
the like.
[0144] In certain embodiments, the ArchT protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 27.
GtR3
[0145] In some embodiments, the light-responsive polypeptide is
responsive to blue light and is a proton pump protein derived from
Guillardia theta, wherein the proton pump protein is capable of
mediating a hyperpolarizing current in the cell when the cell is
illuminated with blue light; such a protein is referred to herein
as a "GtR3 protein" or a "GtR3 polypeptide". The light can have a
wavelength between about 450 and about 495 nm or can have a
wavelength of about 490 nm. In some embodiment, a GtR3 protein
comprises an amino acid sequence at least 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence
shown in SEQ ID NO: 28 (GtR3). The GtR3 protein can additionally
comprise substitutions, deletions, and/or insertions introduced
into a native amino acid sequence to increase or decrease
sensitivity to light, increase or decrease sensitivity to
particular wavelengths of light, and/or increase or decrease the
ability of the GtR3 protein to regulate the polarization state of
the plasma membrane of the cell. Additionally, the GtR3 protein can
comprise one or more conservative amino acid substitutions and/or
one or more non-conservative amino acid substitutions. The GtR3
protein containing substitutions, deletions, and/or insertions
introduced into the native amino acid sequence suitably retains the
ability to hyperpolarize the plasma membrane of a neuronal cell in
response to light.
[0146] In some cases, a GtR3 protein comprises a core amino acid
sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 28
and at least one (such as one, two, three, or more) amino acid
sequence motifs which enhance transport to the plasma membranes of
mammalian cells selected from the group consisting of a signal
peptide, an ER export signal, and a membrane trafficking signal. In
some embodiments, GtR3 protein comprises an N-terminal signal
peptide and a C-terminal ER export signal. In some embodiments, the
GtR3 protein comprises an N-terminal signal peptide and a
C-terminal trafficking signal. In some embodiments, the
light-responsive proton pump protein comprises an N-terminal signal
peptide, a C-terminal ER Export signal, and a C-terminal
trafficking signal. In some embodiments, the GtR3 protein comprises
a C-terminal ER Export signal and a C-terminal trafficking signal.
In some embodiments, the signal peptide comprises the amino acid
sequence MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO: 62). In some
embodiments, the first 19 amino acids are replaced with
MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO: 62). In some embodiments,
the C-terminal ER Export signal and the C-terminal trafficking
signal are linked by a linker. The linker can be any of about 5,
10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,
300, 400, or 500 amino acids in length. The GtR3 protein may
further comprise a fluorescent protein, for example, but not
limited to, a yellow fluorescent protein, a red fluorescent
protein, a green fluorescent protein, or a cyan fluorescent
protein. In some embodiments the ER Export signal is more
C-terminally located than the trafficking signal. In some
embodiments the trafficking signal is more C-terminally located
than the ER Export signal.
[0147] In some embodiments, the trafficking signal is derived from
the amino acid sequence of the human inward rectifier potassium
channel Kir2.1. In other embodiments, the trafficking signal
comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56). Trafficking sequences that are suitable for use can comprise
an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100%, amino acid sequence identity
to an amino acid sequence such a trafficking sequence of human
inward rectifier potassium channel Kir2.1 (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In some cases, the ER export
signal is, e.g., VXXSL (where X is any amino acid) (e.g., VKESL
(SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.); NANSFCYENEVALTSK
(SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X is any amino
acid), e.g., FCYENEV (SEQ ID NO: 61); and the like.
[0148] In certain embodiments, a GtR3 protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 29.
Oxy
[0149] In some embodiments, a light-activated protein is an
Oxyrrhis marina (Oxy) proton pump that can transport one or more
protons across the plasma membrane of a cell when the cell is
illuminated with light. The light can have a wavelength between
about 500 and about 560 nm or can have a wavelength of about 530
nm. In some embodiments, the Oxy protein comprises an amino acid
sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO: 30. The Oxy protein can additionally comprise substitutions,
deletions, and/or insertions introduced into a native amino acid
sequence to increase or decrease sensitivity to light, increase or
decrease sensitivity to particular wavelengths of light, and/or
increase or decrease the ability of the Oxy protein to transport
ions across the plasma membrane of a target cell. Additionally, the
Oxy protein can comprise one or more conservative amino acid
substitutions and/or one or more non-conservative amino acid
substitutions. The Oxy protein containing substitutions, deletions,
and/or insertions introduced into the native amino acid sequence
suitably retains the ability to transport ions across the plasma
membrane of a target cell in response to light.
[0150] In some embodiments, an Oxy protein comprises at least one
(such as one, two, three, or more) amino acid sequence motifs that
enhance transport to the plasma membranes of target cells selected
from the group consisting of a signal peptide, an ER export signal,
and a membrane trafficking signal. In some embodiments, the Oxy
protein comprises an N-terminal signal peptide and a C-terminal ER
export signal. In some embodiments, the Oxy protein includes an
N-terminal signal peptide and a C-terminal trafficking signal. In
some embodiments, the Oxy protein comprises an N-terminal signal
peptide, a C-terminal ER export signal, and a C-terminal
trafficking signal. In some embodiments, the Oxy protein comprises
a C-terminal ER export signal and a C-terminal trafficking signal.
In some embodiments, the C-terminal ER export signal and the
C-terminal trafficking signal are linked by a linker. The linker
can be any of about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175,
200, 225, 250, 275, 300, 400, or 500 amino acids in length. The Oxy
protein may further comprise a fluorescent protein, for example,
but not limited to, a yellow fluorescent protein, a red fluorescent
protein, a green fluorescent protein, or a cyan fluorescent
protein. In some embodiments the ER export signal is more
C-terminally located than the trafficking signal. In some
embodiments the trafficking signal is more C-terminally located
than the ER Export signal.
[0151] In some cases, the Oxy polypeptide comprises a membrane
trafficking signal and/or an ER export signal. In some embodiments,
the trafficking signal can be derived from the amino acid sequence
of the human inward rectifier potassium channel Kir2.1. In other
embodiments, the trafficking signal comprises the amino acid
sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Trafficking
sequences that are suitable for use can comprise an amino acid
sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%, amino acid sequence identity to an amino
acid sequence such a trafficking sequence of human inward rectifier
potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In some cases, the ER export signal is, e.g., VXXSL (where X
is any amino acid) (e.g., VKESL (SEQ ID NO: 57), VLGSL (SEQ ID NO:
58); etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE (SEQ ID NO:
60) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 61); and
the like.
[0152] In certain embodiments, the Oxy protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 31.
Mac
[0153] In some embodiments, the light-responsive proton pump
protein (referred to herein as "Mac protein") is responsive to
light and is derived from Leptosphaeria maculans, wherein the Mac
proton pump protein is capable of pumping protons across the
membrane of a cell when the cell is illuminated with 520 nm to 560
nm light. The light can have a wavelength between about 520 nm to
about 560 nm. In some cases, a Mac protein comprises an amino acid
sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 32
or SEQ ID NO: 33 (Mac; Mac 3.0). The Mac protein can additionally
comprise substitutions, deletions, and/or insertions introduced
into a native amino acid sequence to increase or decrease
sensitivity to light, increase or decrease sensitivity to
particular wavelengths of light, and/or increase or decrease the
ability of the Mac protein to regulate the polarization state of
the plasma membrane of the cell. Additionally, the Mac protein can
comprise one or more conservative amino acid substitutions and/or
one or more non-conservative amino acid substitutions. A Mac
protein containing substitutions, deletions, and/or insertions
introduced into the native amino acid sequence suitably retains the
ability to pump protons across the plasma membrane of a neuronal
cell in response to light.
[0154] In other aspects, a Mac protein comprises a core amino acid
sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 32
and at least one (such as one, two, three, or more) amino acid
sequence motifs which enhance transport to the plasma membranes of
mammalian cells selected from the group consisting of a signal
peptide, an ER export signal, and a membrane trafficking signal. In
some embodiments, the Mac protein comprises an N-terminal signal
peptide and a C-terminal ER export signal. In some embodiments, the
Mac protein comprises an N-terminal signal peptide and a C-terminal
trafficking signal. In some embodiments, the Mac protein comprises
an N-terminal signal peptide, a C-terminal ER Export signal, and a
C-terminal trafficking signal. In some embodiments, the Mac protein
comprises a C-terminal ER Export signal and a C-terminal
trafficking signal. In some embodiments, the C-terminal ER Export
signal and the C-terminal trafficking signal are linked by a
linker. The linker can comprise any of about 5, 10, 20, 30, 40, 50,
75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. The Mac protein may further comprise a fluorescent
protein, for example, but not limited to, a yellow fluorescent
protein, a red fluorescent protein, a green fluorescent protein, or
a cyan fluorescent protein. In some embodiments the ER Export
signal is more C-terminally located than the trafficking signal. In
some embodiments the trafficking signal is more C-terminally
located than the ER Export signal.
[0155] In some cases, the Mac polypeptide includes a membrane
trafficking signal and/or an ER export signal. In some embodiments,
the trafficking signal can be derived from the amino acid sequence
of the human inward rectifier potassium channel Kir2.1. In other
embodiments, the trafficking signal comprises the amino acid
sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Trafficking
sequences that are suitable for use can comprise an amino acid
sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%, amino acid sequence identity to an amino
acid sequence such a trafficking sequence of human inward rectifier
potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In some cases, the ER export signal is, e.g., VXXSL (where X
is any amino acid) (e.g., VKESL (SEQ ID NO: 57), VLGSL (SEQ ID NO:
58); etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE (SEQ ID NO:
60) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 61); and
the like.
[0156] Further disclosure related to light-activated proton pump
proteins can be found in International Patent Application No.
PCT/US2011/028893, the disclosure of which is hereby incorporated
by reference in its entirety.
NpHR
[0157] In some cases, a suitable light-responsive chloride pump
protein is derived from Natronomonas pharaonis; such a protein is
referred to herein as an "NpHR protein" or an "NpHR polypeptide."
In some embodiments, the NpHR protein can be responsive to amber
light as well as red light and can mediate a hyperpolarizing
current in the neuron when the NpHR protein is illuminated with
amber or red light. The wavelength of light that can activate the
NpHR protein can be between about 580 and 630 nm. In some
embodiments, the light can be at a wavelength of about 589 nm or
the light can have a wavelength greater than about 630 nm (e.g.
less than about 740 nm). In another embodiment, the light has a
wavelength of around 630 nm. In some embodiments, the NpHR protein
can hyperpolarize a neural membrane for at least about 90 minutes
when exposed to a continuous pulse of light. In some embodiments,
the NpHR protein comprises an amino acid sequence at least about
75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identical to the sequence shown in SEQ ID NO: 34. Additionally, the
NpHR protein can comprise substitutions, deletions, and/or
insertions introduced into a native amino acid sequence to increase
or decrease sensitivity to light, increase or decrease sensitivity
to particular wavelengths of light, and/or increase or decrease the
ability of the NpHR protein to regulate the polarization state of
the plasma membrane of the cell. In some embodiments, the NpHR
protein comprises one or more conservative amino acid
substitutions. In some embodiments, the NpHR protein comprises one
or more non-conservative amino acid substitutions. A NpHR protein
containing substitutions, deletions, and/or insertions introduced
into the native amino acid sequence suitably retains the ability to
hyperpolarize the plasma membrane of a neuronal cell in response to
light.
[0158] In some cases, an NpHR protein comprises a core amino acid
sequence at least about 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO: 34; and an endoplasmic reticulum (ER) export signal. This ER
export signal can be fused to the C-terminus of the core amino acid
sequence or can be fused to the N-terminus of the core amino acid
sequence. In some embodiments, the ER export signal is linked to
the core amino acid sequence by a linker. The linker can be any of
about 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250,
275, 300, 400, or 500 amino acids in length. The linker may further
comprise a fluorescent protein, for example, but not limited to, a
yellow fluorescent protein, a red fluorescent protein, a green
fluorescent protein, or a cyan fluorescent protein. In some
embodiments, the ER export signal comprises the amino acid sequence
FXYENE (SEQ ID NO: 60), where X can be any amino acid. In another
embodiment, the ER export signal comprises the amino acid sequence
VXXSL, where X can be any amino acid. In some embodiments, the ER
export signal comprises the amino acid sequence FCYENEV (SEQ ID NO:
61).
[0159] Endoplasmic reticulum (ER) export sequences that are
suitable for use include, e.g., VXXSL (where X is any amino acid))
(e.g., VKESL (SEQ ID NO: 57), VLGSL (SEQ ID NO: 58); etc.);
NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE (SEQ ID NO: 60) (where X
is any amino acid), e.g., FCYENEV (SEQ ID NO: 61); and the like. An
ER export sequence can have a length of from about 5 amino acids to
about 25 amino acids, e.g., from about 5 amino acids to about 10
amino acids, from about 10 amino acids to about 15 amino acids,
from about 15 amino acids to about 20 amino acids, or from about 20
amino acids to about 25 amino acids.
[0160] In other aspects, an NpHR protein comprises core amino acid
sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 34
and a trafficking signal (e.g., which can enhance transport of the
NpHR protein to the plasma membrane). The trafficking signal may be
fused to the C-terminus of the core amino acid sequence or may be
fused to the N-terminus of the core amino acid sequence. In some
embodiments, the trafficking signal can be linked to the core amino
acid sequence by a linker, which can be any of about 5, 10, 20, 30,
40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or
500 amino acids in length. The NpHR protein may further comprise a
fluorescent protein, for example, but not limited to, a yellow
fluorescent protein, a red fluorescent protein, a green fluorescent
protein, or a cyan fluorescent protein. In some embodiments, the
trafficking signal can be derived from the amino acid sequence of
the human inward rectifier potassium channel Kir2.1. In other
embodiments, the trafficking signal can comprise the amino acid
sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56).
[0161] In some aspects, an NpHR protein comprises a core amino acid
sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 34;
and at least one (such as one, two, three, or more) amino acid
sequence motifs which enhance transport to the plasma membranes of
mammalian cells selected from the group consisting of an ER export
signal, a signal peptide, and a membrane trafficking signal. In
some embodiments, the NpHR protein includes an N-terminal signal
peptide, a C-terminal ER Export signal, and a C-terminal
trafficking signal. In some embodiments, the C-terminal ER Export
signal and the C-terminal trafficking signal are linked by a
linker. The linker can be any of about 5, 10, 20, 30, 40, 50, 75,
100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino
acids in length. The NpHR protein can also further comprise a
fluorescent protein, for example, but not limited to, a yellow
fluorescent protein, a red fluorescent protein, a green fluorescent
protein, or a cyan fluorescent protein. In some embodiments the ER
Export signal can be more C-terminally located than the trafficking
signal. In other embodiments the trafficking signal is more
C-terminally located than the ER Export signal. In some
embodiments, the signal peptide includes the amino acid sequence
MTETLPPVTESAVALQAE (SEQ ID NO: 66). In another embodiment, the NpHR
protein includes an amino acid sequence at least 95% identical to
SEQ ID NO: 35. In another embodiment, the NpHR protein includes an
amino acid sequence at least 95% identical to SEQ ID NO: 36.
[0162] Moreover, in other aspects, an NpHR protein a core amino
acid sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID
NO: 34, wherein the N-terminal signal peptide of SEQ ID NO: 34 is
deleted or substituted. In some embodiments, other signal peptides
(such as signal peptides from other opsins) can be used. The
light-responsive protein can further comprise an ER transport
signal and/or a membrane trafficking signal described herein.
[0163] In some embodiments, the light-responsive protein is an NpHR
protein that comprises an amino acid sequence at least 75%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% identical to the sequence shown
in SEQ ID NO: 34. In some embodiments, the NpHR protein further
comprises an endoplasmic reticulum (ER) export signal and/or a
membrane trafficking signal. For example, the NpHR protein
comprises an amino acid sequence at least 95% identical to the
sequence shown in SEQ ID NO: 34 and an endoplasmic reticulum (ER)
export signal. In some embodiments, the amino acid sequence at
least 95% identical to the sequence shown in SEQ ID NO: 34 is
linked to the ER export signal through a linker. In some
embodiments, the ER export signal comprises the amino acid sequence
FXYENE (SEQ ID NO: 60), where X can be any amino acid. In another
embodiment, the ER export signal comprises the amino acid sequence
VXXSL, where X can be any amino acid. In some embodiments, the ER
export signal comprises the amino acid sequence FCYENEV (SEQ ID NO:
61). In some embodiments, the NpHR protein comprises an amino acid
sequence at least 95% identical to the sequence shown in SEQ ID NO:
34, an ER export signal, and a membrane trafficking signal. In
other embodiments, the NpHR protein comprises, from the N-terminus
to the C-terminus, the amino acid sequence at least 95% identical
to the sequence shown in SEQ ID NO: 34, the ER export signal, and
the membrane trafficking signal. In other embodiments, the NpHR
protein comprises, from the N-terminus to the C-terminus, the amino
acid sequence at least 95% identical to the sequence shown in SEQ
ID NO: 34, the membrane trafficking signal, and the ER export
signal. In some embodiments, the membrane trafficking signal is
derived from the amino acid sequence of the human inward rectifier
potassium channel Kir2.1. In some embodiments, the membrane
trafficking signal comprises the amino acid sequence
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). In some embodiments, the
membrane trafficking signal is linked to the amino acid sequence at
least 95% identical to the sequence shown in SEQ ID NO: 34 by a
linker. In some embodiments, the membrane trafficking signal is
linked to the ER export signal through a linker. The linker may be
any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225,
250, 275, 300, 400, or 500 amino acids in length. The linker may
further comprise a fluorescent protein, for example, but not
limited to, a yellow fluorescent protein, a red fluorescent
protein, a green fluorescent protein, or a cyan fluorescent
protein. In some embodiments, the light-responsive protein further
comprises an N-terminal signal peptide.
[0164] Further disclosure related to light-responsive chloride pump
proteins can be found in U.S. Patent Application Publication Nos:
2009/0093403 and 2010/0145418 as well as in International Patent
Application NO: PCT/US201 1/028 893, the disclosures of each of
which are hereby incorporated by reference in their entireties.
Dunaliella salina Light-Responsive Polypeptide
[0165] In some embodiments, a suitable light-responsive ion channel
protein is, e.g., a DsChR protein derived from Dunaliella salina,
wherein the ion channel protein is capable of mediating a
hyperpolarizing current in the cell when the cell is illuminated
with light. The light can have a wavelength between about 470 nm
and about 510 nm or can have a wavelength of about 490 nm. In some
embodiments, a DsChR protein comprises an amino acid sequence at
least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
or 100% identical to the sequence shown in SEQ ID NO: 37. The DsChR
protein can additionally comprise substitutions, deletions, and/or
insertions introduced into a native amino acid sequence to increase
or decrease sensitivity to light, increase or decrease sensitivity
to particular wavelengths of light, and/or increase or decrease the
ability of the DsChR protein to regulate the polarization state of
the plasma membrane of the cell. Additionally, the DsChR protein
can comprise one or more conservative amino acid substitutions
and/or one or more non-conservative amino acid substitutions. A
DsChR protein containing substitutions, deletions, and/or
insertions introduced into the native amino acid sequence suitably
retains the ability to transport ions across the plasma membrane of
a neuronal cell in response to light.
[0166] In some case, a DsChR protein comprises a core amino acid
sequence at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO: 37;
and at least one (such as one, two, three, or more) amino acid
sequence motifs which enhance transport to the plasma membranes of
mammalian cells selected from the group consisting of a signal
peptide, an ER export signal, and a membrane trafficking signal. In
some embodiments, the DsChR protein comprises an N-terminal signal
peptide and a C-terminal ER export signal. In some embodiments, the
DsChR protein comprises an N-terminal signal peptide and a
C-terminal trafficking signal. In some embodiments, the DsChR
protein comprises an N-terminal signal peptide, a C-terminal ER
Export signal, and a C-terminal trafficking signal. In some
embodiments, the DsChR protein comprises a C-terminal ER Export
signal and a C-terminal trafficking signal. In some embodiments,
the C-terminal ER Export signal and the C-terminal trafficking
signal are linked by a linker. The linker can be any of about 5,
10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275,
300, 400, or 500 amino acids in length. The DsChR protein may
further comprise a fluorescent protein, for example, but not
limited to, a yellow fluorescent protein, a red fluorescent
protein, a green fluorescent protein, or a cyan fluorescent
protein. In some embodiments the ER Export signal is more
C-terminally located than the trafficking signal. In some
embodiments the trafficking signal is more C-terminally located
than the ER Export signal.
[0167] In some cases, the DsChR polypeptide comprises a membrane
trafficking signal and/or an ER export signal. In some embodiments,
the trafficking signal is derived from the amino acid sequence of
the human inward rectifier potassium channel Kir2.1. In other
embodiments, the trafficking signal comprises the amino acid
sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56). Trafficking
sequences that are suitable for use can comprise an amino acid
sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100%, amino acid sequence identity to an amino
acid sequence such a trafficking sequence of human inward rectifier
potassium channel Kir2.1 (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In some cases, the ER export signal is, e.g., VXXSL (where X
is any amino acid) (e.g., VKESL (SEQ ID NO: 57), VLGSL (SEQ ID NO:
58); etc.); NANSFCYENEVALTSK (SEQ ID NO: 59); FXYENE (SEQ ID NO:
60) (where X is any amino acid), e.g., FCYENEV (SEQ ID NO: 61); and
the like.
[0168] In certain embodiments, the DsChR protein comprises an amino
acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in
SEQ ID NO: 38.
Anion Channel Polypeptides Based on C1C2
[0169] In some embodiments, a light-responsive anion channel
polypeptide is a C1C2 protein. In some embodiments, a C1C2
polypeptide comprises an amino acid sequence having at least 58%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth in SEQ ID NO: 12. In some embodiments, the amino
acid sequence of the C1C2 protein is modified by introducing one or
more of the following mutations into the amino acid sequence: T98S,
E129S, E140S, E162S, V156K, H173R, T285N, V281K and/or N297Q. In
some embodiments, a C1C2 protein comprises the amino acid sequence
of the protein C1C2 with all 9 of the above-listed amino acid
substitutions, such that the amino acid sequence of the C1C2
polypeptide is that set forth in SEQ ID NO: 39.
[0170] In some embodiments, a C1C2 polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 39; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid
substitutions selected from T98S, E129S, E140S, E162S, V156K,
H173R, T285N, V281K and/or N297Q, relative to the amino acid
sequence of C1C2 (SEQ ID NO: 12). In some embodiments, a C1C2
polypeptide includes an amino acid sequence having at least 58%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth in SEQ ID NO: 39; and includes T98S, E129S,
E140S, E162S, and T285N substitutions relative to the amino acid
sequence of C1C2. In some embodiments, a C1C2 polypeptide includes
an amino acid sequence having at least 58%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino
acid sequence identity to the amino acid sequence set forth in SEQ
ID NO: 39; and includes V156K, H173R, V281K, and N297Q
substitutions relative to the amino acid sequence of C1C2.
[0171] In some embodiments, a C1C2 polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 39; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129,
S140, S162, K156, R173, N285, K281, and Q297, where the amino acid
numbering is as set forth in SEQ ID NO: 39. In some embodiments, a
C1C2 polypeptide comprises an amino acid sequence having at least
58%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%,
at least 99%, or 100%, amino acid sequence identity to the amino
acid sequence set forth in SEQ ID NO: 39; and includes S98, S129,
S140, S162, K156, R173, N285, K281, and Q297, where the amino acid
numbering is as set forth in SEQ ID NO: 39. In any one of these
embodiments, a C1C2 polypeptide can comprise a membrane trafficking
signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of
these embodiments, a C1C2 polypeptide can comprise an ER export
signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of these
embodiments, a C1C2 polypeptide comprises both a membrane
trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and
an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). Thus, in
certain embodiments, the C1C2 protein comprises an amino acid
sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO: 40.
[0172] In some embodiments, a C1C2 polypeptide is based on the
amino acid sequence of the protein C1C2 (SEQ ID NO: 12), wherein
the amino acid sequence has been modified by replacing the first 50
N-terminal amino acids of C1C2 with amino acids 1-11 from the
protein ChR2 (MDYGGALSAVG) (SEQ ID NO: 55). In some embodiments, a
suitable light-responsive anion channel polypeptide is referred to
as "ibC1C2" and comprises an amino acid sequence having at least
58%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%,
at least 99%, or 100%, amino acid sequence identity to the amino
acid sequence set forth in SEQ ID NO: 43; and includes 1, 2, 3, 4,
5, 6, 7, 8, or 9 of: S59, S90, S101, S123, K117, R134, N246, K242,
and Q258, where the amino acid numbering is as set forth in SEQ ID
NO: 43. In some embodiments, a suitable light-responsive anion
channel polypeptide comprises an amino acid sequence having at
least 58%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
98%, at least 99%, or 100%, amino acid sequence identity to the
amino acid sequence set forth in SEQ ID NO: 43; and includes S59,
S90, S101, S123, K117, R134, N246, K242, and Q258, where the amino
acid numbering is as set forth in SEQ ID NO: 43. In some
embodiments, a suitable light-responsive anion channel polypeptide
comprises the amino acid sequence set forth in SEQ ID NO: 43. In
any one of these embodiments, a suitable anion channel polypeptide
comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV
(SEQ ID NO: 56)). In any one of these embodiments, a suitable anion
channel polypeptide comprises an ER export signal (e.g., FCYENEV
(SEQ ID NO: 61)). In any one of these embodiments, a suitable anion
channel polypeptide comprises both a membrane trafficking signal
(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export
signal (e.g., FCYENEV (SEQ ID NO: 61)). Thus, in certain
embodiments, the ibC1C2 protein comprises an amino acid sequence
that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:
44.
[0173] In some embodiments, a suitable light-responsive anion
channel polypeptide is based on the amino acid sequence of the
protein C1C2 (SEQ ID NO: 12), wherein the cysteine amino acid
residue at position 167 has been replaced by a threonine residue.
In some embodiments, a suitable light-responsive anion channel
polypeptide, e.g., SwiChRc.sub.T, comprises an amino acid sequence
having at least 58%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98%, at least 99%, or 100%, amino acid sequence identity
to the amino acid sequence set forth in SEQ ID NO: 41; and
comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162,
K156, R173, N285, K281, and Q297; and includes T167. In some
embodiments, a suitable light-responsive anion channel polypeptide
comprises an amino acid sequence having at least 58%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 98%, at least 99%, or
100%, amino acid sequence identity to the amino acid sequence set
forth SEQ ID NO: 41; and includes S98, S129, S140, S162, K156,
R173, N285, K281, and Q297; and includes T167, where the amino acid
numbering is as set forth in SEQ ID NO: 41. In some embodiments, a
light-responsive anion channel polypeptide comprises the amino acid
sequence provided in SEQ ID NO: 5. In some of these embodiments,
the light-responsive polypeptide exhibits prolonged stability of
photocurrents. In some embodiments, the first 50 amino acids are
replaced with MDYGGALSAVG (SEQ ID NO: 55). In any one of these
embodiments, a suitable anion channel polypeptide comprises a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In any one of these embodiments, a suitable anion channel
polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID
NO: 61)). In any one of these embodiments, a suitable anion channel
polypeptide comprises both a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal
(e.g., FCYENEV (SEQ ID NO: 61)).
[0174] In some embodiments, a suitable light-responsive anion
channel polypeptide is based on the amino acid sequence of the
protein C1C2, wherein the cysteine amino acid residue at position
167 has been replaced by an alanine residue. In some embodiments, a
suitable light-responsive anion channel polypeptide, SwiChRC.sub.A,
comprises an amino acid sequence having at least 58%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 98%, at least 99%, or
100%, amino acid sequence identity to the amino acid sequence set
forth SEQ ID NO: 41; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of:
S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and
includes A167, where the amino acid numbering is as set forth in
SEQ ID NO: 41. In some embodiments, a suitable light-responsive
anion channel polypeptide comprises an amino acid sequence having
at least 58%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, at least 99%, or 100%, amino acid sequence identity to
the amino acid sequence set forth SEQ ID NO: 41; and includes S98,
S129, S140, S162, K156, R173, N285, K281, and Q297; and includes
A167, where the amino acid numbering is as set forth in SEQ ID NO:
41. In some embodiments, the first 50 amino acids are replaced with
MDYGGALSAVG (SEQ ID NO: 55). In any one of these embodiments, a
suitable anion channel polypeptide comprises a membrane trafficking
signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of
these embodiments, a subject anion channel polypeptide includes an
ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of
these embodiments, a subject anion channel polypeptide comprises
both a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ
ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:
61)).
[0175] In some embodiments, a suitable light-responsive anion
channel polypeptide is based on the amino acid sequence of the
protein C1C2, wherein the cysteine amino acid residue at position
167 has been replaced by a serine residue. In some embodiments, a
suitable light-responsive anion channel polypeptide, SwiChRcs,
comprises an amino acid sequence having at least 58%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, at least 98%, at least 99%, or
100%, amino acid sequence identity to the amino acid sequence set
forth SEQ ID NO: 41; and includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of:
S98, S129, S140, S162, K156, R173, N285, K281, and Q297; and
includes S167, where the amino acid numbering is as set forth in
SEQ ID NO: 41. In some embodiments, a suitable light-responsive
anion channel polypeptide comprises an amino acid sequence having
at least 58%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, at least 99%, or 100%, amino acid sequence identity to
the amino acid sequence set forth SEQ ID NO: 41; and includes S98,
S129, S140, S162, K156, R173, N285, K281, and Q297; and includes
S167, where the amino acid numbering is as set forth in SEQ ID NO:
41. In some embodiments, the first 50 amino acids are replaced with
MDYGGALSAVG (SEQ ID NO: 55). In any one of these embodiments, a
suitable anion channel polypeptide comprises a membrane trafficking
signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of
these embodiments, a subject anion channel polypeptide includes an
ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of
these embodiments, a subject anion channel polypeptide comprises
both a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ
ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:
61)).
[0176] In certain embodiments, the SwiChR protein comprises an
amino acid sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence
shown in SEQ ID NO: 42.
[0177] In some embodiments, a suitable light-responsive anion
channel polypeptide, SwiChR, comprises an amino acid sequence
having at least 58%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98%, at least 99%, or 100%, amino acid sequence identity
to the amino acid sequence set forth SEQ ID NO: 41; and includes 1,
2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162, K156, R173,
N285, K281, and Q297; includes N195, or A195; and includes A167,
where the amino acid numbering is as set forth in SEQ ID NO: 41. In
some embodiments, a suitable light-responsive anion channel
polypeptide comprises an amino acid sequence having at least 58%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth SEQ ID NO: 41; and includes S98, S129, S140,
S162, K156, R173, N285, K281, and Q297; includes A167; and includes
N195, or A195, where the amino acid numbering is as set forth in
SEQ ID NO: 41. In some embodiments, the first 50 amino acids are
replaced with MDYGGALSAVG (SEQ ID NO: 55). In any one of these
embodiments, a subject anion channel polypeptide comprises a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In any one of these embodiments, a subject anion channel
polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID
NO: 61)). In any one of these embodiments, a subject anion channel
polypeptide comprises both a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal
(e.g., FCYENEV (SEQ ID NO: 61)).
[0178] In some embodiments, a suitable light-responsive anion
channel polypeptide is based on the amino acid sequence of the
protein C1C2 with one or more of the modifications described above,
wherein the aspartate amino acid residue at original position 195
has been replaced by an alanine residue. In certain embodiments
wherein the first 50 N-terminal amino acids of the protein are
replaced by amino acids 1-11 from the protein ChR2, the aspartate
amino acid residue at position 156 (which corresponds to original
position 195 of the C1C2 amino acid sequence set forth in SEQ ID
NO: 12) is replaced by an alanine residue.
[0179] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein C1C2 with one or more of the modifications described
above, wherein the aspartate amino acid residue at original
position 195 has been replaced by an asparagine residue. In certain
embodiments wherein the first 50 N-terminal amino acids of the
protein are replaced by amino acids 1-11 from the protein ChR2, the
aspartate amino acid residue at position 156 (which corresponds to
original position 195 of the C1C2 amino acid sequence set forth in
SEQ ID NO: 12) is replaced by an asparagine residue.
[0180] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide comprises an amino acid sequence
having at least 58%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98%, at least 99%, or 100%, amino acid sequence identity
to the amino acid sequence set forth in SEQ ID NO: 43; and includes
1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123, K117, R134,
N246, K242, and Q258; and includes A128, T128 or S128, where the
amino acid numbering is as set forth in SEQ ID NO: 43. In some
embodiments, a suitable hyperpolarizing light-responsive
polypeptide comprises an amino acid sequence having at least 58%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth in SEQ ID NO: 43; and includes S59, S90, S101,
S123, K117, R134, N246, K242, and Q258; and includes A128, T128 or
S128, where the amino acid numbering is as set forth in SEQ ID NO:
43. In any one of these embodiments, a subject anion channel
polypeptide comprises a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of these
embodiments, a suitable anion channel polypeptide comprises an ER
export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of these
embodiments, a suitable anion channel polypeptide includes both a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)).
Anion Channel Proteins Based on ChR2
[0181] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein ChR2. The amino acid sequence of ChR2 is set forth in
SEQ ID NO: 4. In some embodiments, the amino acid sequence of the
ChR2 protein has been modified by introducing one or more of the
following mutations into the amino acid sequence: A59S, E90S,
E101S, E123S, Q117K, H134R, V242K, T246N and/or N258Q. In some
embodiments, a suitable hyperpolarizing light-responsive
polypeptide comprises the amino acid sequence of the protein ChR2
with all 9 of the above-listed amino acid substitutions, such that
the amino acid sequence of the polypeptide is provided in SEQ ID
NO: 45 (iChR2).
[0182] In some embodiments, a suitable light-responsive anion
channel polypeptide iChR2 comprises an amino acid sequence having
at least 58%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, at least 99%, or 100%, amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO: 45; and includes 1,
2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from
A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N and/or N258Q,
relative to the amino acid sequence of ChR2 (SEQ ID NO: 4).
[0183] In some embodiments, a suitable light-responsive polypeptide
("iChR2") comprises an amino acid sequence having at least 58%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth in SEQ ID NO: 45; and includes 1, 2, 3, 4, 5, 6,
7, 8, or 9 of: S59, S90, S101, S123, K117, R134, K242, N246 and
Q258, where the amino acid numbering is as set forth in SEQ ID NO:
45. In some embodiments, an iChR2 polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 45; and includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of: S59,
S90, S101, S123, K117, R134, K242, N246, Q258, and either N156 or
A156, and either T128, A128, or S128, where the amino acid
numbering is as set forth in SEQ ID NO: 45. In some embodiments, an
iChR2 polypeptide comprises an amino acid sequence having at least
58%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%,
at least 99%, or 100%, amino acid sequence identity to the amino
acid sequence set forth in SEQ ID NO: 45; and includes S59, S90,
S101, S123, K117, R134, K242, N246 and Q258, where the amino acid
numbering is as set forth in SEQ ID NO: 45. In any one of these
embodiments, an iChR2 polypeptide can comprise a membrane
trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In
any one of these embodiments, an iChR2 polypeptide can comprise an
ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of
these embodiments, an iChR2 polypeptide can comprise both a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). Thus
in certain embodiments, the iChR2 protein comprises an amino acid
sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO: 46.
Anion Channel Polypeptides Based on C1V1
[0184] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein C1V1. The amino acid sequence of C1V1 is set forth in
SEQ ID NO: 10. In some embodiments, the amino acid sequence of the
C1V1 protein has been modified by introducing one or more of the
following mutations into the amino acid sequence: T98S, E129S,
E140S, E162S, V156K, H173R, A285N, P281K and/or N297Q. In some
embodiments, a hyperpolarizing light-responsive polypeptide
comprises the amino acid sequence of the protein C1V1 with all 9 of
the above-listed amino acid substitutions, such that the amino acid
sequence of the polypeptide is provided in SEQ ID NO: 47.
[0185] In some embodiments, a suitable light-responsive anion
channel polypeptide, iC1V1, comprises an amino acid sequence having
at least 58%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, at least 99%, or 100%, amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO: 47; and includes 1,
2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from
T98S, E129S, E140S, E162S, V156K, H173R, A285N, P281K and/or N297Q,
relative to the amino acid sequence of C1V1 (SEQ ID NO: 10).
[0186] In some embodiments, a suitable light-responsive anion
channel polypeptide, iC1V1, comprises an amino acid sequence having
at least 58%, at least 60%, at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 98%, at least 99%, or 100%, amino acid sequence identity to
the amino acid sequence set forth in SEQ ID NO: 47; and includes 1,
2, 3, 4, 5, 6, 7, 8, or 9 of: S98, S129, S140, S162, K156, R173,
N285, K281, and Q297, where the amino acid numbering is as set
forth in SEQ ID NO: 47. In some embodiments, a suitable
light-responsive anion channel polypeptide (referred to as
"iC1V1"), comprises an amino acid sequence having at least 58%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth in SEQ ID NO: 47; and includes 1, 2, 3, 4, 5, 6,
7, 8, or 9 of: S98, S129, S140, S162, K156, R173, N285, K281, and
Q297, and includes N195, where the amino acid numbering is as set
forth in SEQ ID NO: 47. In some embodiments, a suitable
light-responsive anion channel polypeptide comprises an amino acid
sequence having at least 58%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to the amino acid sequence set forth in SEQ ID NO: 47; and
includes S98, S129, S140, S162, K156, R173, N285, K281, and Q297,
where the amino acid numbering is as set forth in SEQ ID NO: 47. In
any one of these embodiments, a suitable anion channel polypeptide
includes a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV
(SEQ ID NO: 56)). In any one of these embodiments, a subject anion
channel polypeptide includes an ER export signal (e.g., FCYENEV
(SEQ ID NO: 61)). In any one of these embodiments, a suitable anion
channel polypeptide comprises both a membrane trafficking signal
(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export
signal (e.g., FCYENEV (SEQ ID NO: 61)). Thus in certain
embodiments, the iC1V1protein can have an amino acid sequence that
is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100% identical to the sequence shown in SEQ ID NO: 48.
[0187] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein C1V1 (SEQ ID NO: 10), wherein the amino acid sequence
has been modified by replacing the first 50 N-terminal amino acids
of C1V1 with amino acids 1-11 from the protein ChR2 (MDYGGALSAVG)
(SEQ ID NO: 55). In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide, ibC1V1, comprises an amino acid
sequence having at least 58%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to the amino acid sequence set forth in SEQ ID NO: 49; and
includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123,
K117, R134, N246, K242, and Q258, where the amino acid numbering is
as set forth in SEQ ID NO: 49. In some embodiments, a suitable
hyperpolarizing light-responsive polypeptide (referred to as
"ibC1V1"), comprises an amino acid sequence having at least 58%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%,
at least 85%, at least 90%, at least 95%, at least 98%, at least
99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth in SEQ ID NO: 49; and includes 1, 2, 3, 4, 5, 6,
7, 8, or 9 of: S59, S90, S101, S123, K117, R134, N246, K242, and
Q258, and includes N156, where the amino acid numbering is as set
forth in SEQ ID NO: 49. In some embodiments, a suitable
hyperpolarizing light-responsive polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 49; and includes S59, S90, S101, S123, K117, R134, N246, K242,
and Q258, where the amino acid numbering is as set forth in SEQ ID
NO: 49. In some embodiments, a suitable light-responsive anion
channel polypeptide comprises the amino acid sequence set forth in
SEQ ID NO: 49. In any one of these embodiments, a suitable anion
channel polypeptide comprises a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of these
embodiments, a suitable anion channel polypeptide comprises an ER
export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of these
embodiments, a subject anion channel polypeptide comprises both a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). Thus
in certain embodiments, an ibC1V1 protein comprises an amino acid
sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO: 50.
[0188] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein C1V1 (SEQ ID NO: 10), wherein the cysteine amino acid
residue at position 167 has been replaced by a threonine residue.
In some embodiments, a suitable hyperpolarizing light-responsive
polypeptide comprises an amino acid sequence having at least 58%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth SEQ ID NO: 47; and includes 1, 2, 3, 4, 5, 6, 7,
8, or 9 of: S98, S129, S140, S162, K156, R173, N285, K281, and
Q297; and includes T167. In some embodiments, a suitable
hyperpolarizing light-responsive polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth SEQ ID NO:
47; and includes S98, S129, S140, S162, K156, R173, N285, K281, and
Q297; and includes T167, S167 or A167, where the amino acid
numbering is as set forth in SEQ ID NO: 47. In some embodiments, a
suitable hyperpolarizing light-responsive polypeptide comprises an
amino acid sequence having at least 58%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino
acid sequence identity to the amino acid sequence set forth SEQ ID
NO: 47; and includes S98, S129, S140, S162, K156, R173, N285, K281,
and Q297; includes T167, S167 or A167; and includes A195 or N195,
where the amino acid numbering is as set forth in SEQ ID NO: 47. In
some embodiments, the first 50 amino acids are replaced with
MDYGGALSAVG (SEQ ID NO: 55). In any one of these embodiments, a
suitable hyperpolarizing light-responsive polypeptide comprises a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)). In any one of these embodiments, a suitable hyperpolarizing
light-responsive polypeptidecomprises an ER export signal (e.g.,
FCYENEV (SEQ ID NO: 61)). In any one of these embodiments, a
suitable hyperpolarizing light-responsive polypeptide includes both
a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID
NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:
61)).
[0189] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein C1V1 with one or more of the modifications described
above, wherein the aspartate amino acid residue at original
position 195 has been replaced by an alanine residue. In certain
embodiments wherein the first 50 N-terminal amino acids of the
protein are replaced by amino acids 1-11 from the protein ChR2, the
aspartate amino acid residue at position 156 (which corresponds to
original position 195 of the C1V1 amino acid sequence set forth in
SEQ ID NO: 10) is replaced by an alanine residue.
[0190] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein C1V1 with one or more of the modifications described
above, wherein the aspartate amino acid residue at original
position 195 has been replaced by an asparagine residue. In certain
embodiments wherein the first 50 N-terminal amino acids of the
protein are replaced by amino acids 1-11 from the protein ChR2, the
aspartate amino acid residue at position 156 (which corresponds to
original position 195 of the C1V1 amino acid sequence set forth in
SEQ ID NO: 10) is replaced by an asparagine residue.
[0191] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide, ibC1V1, comprises an amino acid
sequence having at least 58%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to the amino acid sequence set forth in SEQ ID NO: 49; and
includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123,
K117, R134, N246, K242, and Q258; and includes T128, A128, or S128,
where the amino acid numbering is as set forth in SEQ ID NO: 49. In
some embodiments, a suitable hyperpolarizing light-responsive
polypeptide comprises an amino acid sequence having at least 58%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth in SEQ ID NO: 49; and includes S59, S90, S101,
S123, K117, R134, N246, K242, and Q258; and includes T128, A128, or
S128, where the amino acid numbering is as set forth in SEQ ID NO:
49. In any one of these embodiments, a suitable anion channel
polypeptide comprises a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of these
embodiments, a suitable anion channel polypeptide comprises an ER
export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of these
embodiments, a suitable anion channel polypeptide comprises both a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)).
[0192] In some embodiments, a suitable hyperpolarizing
light-responsive polypeptide comprises an amino acid sequence
having at least 58%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98%, at least 99%, or 100%, amino acid sequence identity
to the amino acid sequence set forth in SEQ ID NO: 49; and includes
1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123, K117, R134,
N246, K242, and Q258; and includes T128, A128, or S128; and
includes A156 or N156, where the amino acid numbering is as set
forth in SEQ ID NO: 49. In some embodiments, a suitable
hyperpolarizing light-responsive polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 49; and includes S59, S90, S101, S123, K117, R134, N246, K242,
and Q258; and includes T128, A128, or S128; and includes A156 or
N156, where the amino acid numbering is as set forth in SEQ ID NO:
49. In any one of these embodiments, a suitable hyperpolarizing
light-responsive polypeptide comprises a membrane trafficking
signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of
these embodiments, a suitable hyperpolarizing light-responsive
polypeptide comprises an ER export signal (e.g., FCYENEV (SEQ ID
NO: 61)). In any one of these embodiments, a subject anion channel
polypeptide includes both a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal
(e.g., FCYENEV (SEQ ID NO: 61)).
Anion Channel Polypeptides Based on ReaChR
[0193] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein ReaChR. The amino acid sequence of ReaChR is set forth
in SEQ ID NO: 14. In some embodiments, the amino acid sequence of
the ReaChR protein has been modified by introducing one or more of
the following mutations into the amino acid sequence: T99S, E130S,
E141S, E163S, V157K, H174R, A286N, P282K and/or N298Q. In some
embodiments, a subject hyperpolarizing light-responsive polypeptide
comprises the amino acid sequence of the protein ReaChR with all 9
of the above-listed amino acid substitutions, such that the amino
acid sequence of the polypeptide is provided in SEQ ID NO: 51.
[0194] In some embodiments, a subject light-responsive anion
channel polypeptide comprises an amino acid sequence having at
least 58%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, at least 95%, at least
98%, at least 99%, or 100%, amino acid sequence identity to the
amino acid sequence set forth in SEQ ID NO: 51; and includes 1, 2,
3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from T99S,
E130S, E141S, E163S, V157K, H174R, A286N, P282K and/or N298Q,
relative to the amino acid sequence of ReaChR (SEQ ID NO: 14).
[0195] In some embodiments, a subject light-responsive anion
channel polypeptide, iReaChR, comprises an amino acid sequence
having at least 58%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98%, at least 99%, or 100%, amino acid sequence identity
to the amino acid sequence set forth in SEQ ID NO: 51; and includes
1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130, S141, S163, K157, R174,
N286, K281, and Q298, where the amino acid numbering is as set
forth in SEQ ID NO: 51. In some embodiments, a subject
light-responsive anion channel polypeptide comprises an amino acid
sequence having at least 58%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to the amino acid sequence set forth in SEQ ID NO: 51; and
includes S99, S130, S141, S163, K157, R174, N286, K281, and Q298,
where the amino acid numbering is as set forth in SEQ ID NO: 51. In
any one of these embodiments, a subject anion channel polypeptide
comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV
(SEQ ID NO: 56)). In any one of these embodiments, a subject anion
channel polypeptide comprises an ER export signal (e.g., FCYENEV
(SEQ ID NO: 61)). In any one of these embodiments, a subject anion
channel polypeptide includes both a membrane trafficking signal
(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export
signal (e.g., FCYENEV (SEQ ID NO: 61)). Thus in certain
embodiments, the iReaChR protein comprises an amino acid sequence
that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:
52.
[0196] In some embodiments, a subject light-responsive anion
channel polypeptide, iReaChR, comprises an amino acid sequence
having at least 58%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98%, at least 99%, or 100%, amino acid sequence identity
to the amino acid sequence set forth in SEQ ID NO: 51; and includes
1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130, S141, S163, K157, R174,
N286, K281, and Q298, and includes N196, where the amino acid
numbering is as set forth in SEQ ID NO: 51. In some embodiments, a
subject light-responsive anion channel polypeptide comprises an
amino acid sequence having at least 58%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino
acid sequence identity to the amino acid sequence set forth in SEQ
ID NO: 51; and includes S99, S130, S141, S163, K157, R174, N286,
K281, and Q298, and includes N196, where the amino acid numbering
is as set forth in SEQ ID NO: 51. In any one of these embodiments,
a subject anion channel polypeptide comprises a membrane
trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In
any one of these embodiments, a subject anion channel polypeptide
comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). In
any one of these embodiments, a subject anion channel polypeptide
comprises both a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal
(e.g., FCYENEV (SEQ ID NO: 61)).
[0197] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein ReaChR (SEQ ID NO: 14), wherein the amino acid sequence
has been modified by replacing the first 51 N-terminal amino acids
of ReaChR with amino acids 1-11 from the protein ChR2 (MDYGGALSAVG)
(SEQ ID NO: 55). In some embodiments, a subject hyperpolarizing
light-responsive polypeptide, ibReaChR, comprises an amino acid
sequence having at least 58%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to the amino acid sequence set forth in SEQ ID NO: 53; and
includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123,
K117, R134, N246, K242, and Q258, where the amino acid numbering is
as set forth in SEQ ID NO: 53. In some embodiments, a subject
hyperpolarizing light-responsive polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 53; and includes S59, S90, S101, S123, K117, R134, N246, K242,
and Q258, where the amino acid numbering is as set forth in SEQ ID
NO: 53. In some embodiments, a subject light-responsive anion
channel polypeptide comprises the amino acid sequence set forth in
SEQ ID NO: 53. In any one of these embodiments, a subject anion
channel polypeptide comprises a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of these
embodiments, a subject anion channel polypeptide comprises an ER
export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of these
embodiments, a subject anion channel polypeptide comprises both a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). Thus
in certain embodiments, the ibReaChR protein can have an amino acid
sequence that is at least 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ
ID NO: 54.
[0198] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein ReaChR (SEQ ID NO: 14), wherein the amino acid sequence
has been modified by replacing the first 51 N-terminal amino acids
of ReaChR with amino acids 1-11 from the protein ChR2 (MDYGGALSAVG)
(SEQ ID NO: 55). In some embodiments, a subject hyperpolarizing
light-responsive polypeptide comprises an amino acid sequence
having at least 58%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%,
at least 98%, at least 99%, or 100%, amino acid sequence identity
to the amino acid sequence set forth in SEQ ID NO: 53; and includes
1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123, K117, R134,
N246, K242, and Q258, and includes N156, where the amino acid
numbering is as set forth in SEQ ID NO: 53. In some embodiments, a
subject hyperpolarizing light-responsive polypeptide comprises an
amino acid sequence having at least 58%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino
acid sequence identity to the amino acid sequence set forth in SEQ
ID NO: 53; and includes S59, S90, S101, S123, K117, R134, N246,
K242, and Q258, and includes N156, where the amino acid numbering
is as set forth in SEQ ID NO: 53. In any one of these embodiments,
a subject anion channel polypeptide comprises a membrane
trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In
any one of these embodiments, a subject anion channel polypeptide
comprises an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). In
any one of these embodiments, a subject anion channel polypeptide
comprises both a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export signal
(e.g., FCYENEV (SEQ ID NO: 61)).
[0199] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein ReaChR (SEQ ID NO: 14), wherein the cysteine amino acid
residue at position 168 has been replaced by a threonine residue.
In some embodiments, a subject hyperpolarizing light-responsive
polypeptide comprises an amino acid sequence having at least 58%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth SEQ ID NO: 51; and includes 1, 2, 3, 4, 5, 6, 7,
8, or 9 of: S99, S130, S141, S163, K157, R174, N286, K281, and
Q298; and includes T168, S168 or A168. In some embodiments, a
subject hyperpolarizing light-responsive polypeptide comprises an
amino acid sequence having at least 58%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino
acid sequence identity to the amino acid sequence set forth SEQ ID
NO: 60; and includes S99, S130, S141, S163, K157, R174, N286, K281,
and Q298; and includes T168, S168 or A168, where the amino acid
numbering is as set forth in SEQ ID NO: 51. In some embodiments,
the first 51 amino acids are replaced with MDYGGALSAVG (SEQ ID NO:
55). In any one of these embodiments, a subject anion channel
polypeptide comprises a membrane trafficking signal (e.g.,
KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of these
embodiments, a subject anion channel polypeptide comprises an ER
export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of these
embodiments, a subject anion channel polypeptide comprises both a
membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO:
56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO: 61)).
[0200] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide, iReaChR, comprises an amino acid
sequence having at least 58%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to the amino acid sequence set forth SEQ ID NO: 51; and
includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S99, S130, S141, S163,
K157, R174, N286, K281, and Q298; includes A196 or N196; and
includes T168, S168, or A168, where the amino acid numbering is as
set forth in SEQ ID NO: 51. In some embodiments, a subject
hyperpolarizing light-responsive polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth SEQ ID NO:
51; and includes S99, S130, S141, S163, K157, R174, N286, K281, and
Q298; includes A196 or N196; and includes T168, S168, or A168,
where the amino acid numbering is as set forth in SEQ ID NO: 51. In
some embodiments, the first 51 amino acids are replaced with
MDYGGALSAVG (SEQ ID NO: 55). In any one of these embodiments, a
subject anion channel polypeptide comprises a membrane trafficking
signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)). In any one of
these embodiments, a subject anion channel polypeptide comprises an
ER export signal (e.g., FCYENEV (SEQ ID NO: 61)). In any one of
these embodiments, a subject anion channel polypeptide includes
both a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV (SEQ
ID NO: 56)) and an ER export signal (e.g., FCYENEV (SEQ ID NO:
61)).
[0201] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein ReaChR with one or more of the modifications described
above, wherein the aspartate amino acid residue at original
position 196 has been replaced by an alanine residue. In certain
embodiments wherein the first 51 N-terminal amino acids of the
protein are replaced by amino acids 1-11 from the protein ChR2, the
aspartate amino acid residue at position 156 (which corresponds to
original position 196 of the ReaChR amino acid sequence set forth
in SEQ ID NO: 14) is replaced by an alanine residue.
[0202] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide is based on the amino acid sequence of
the protein ReaChR with one or more of the modifications described
above, wherein the aspartate amino acid residue at original
position 196 has been replaced by an asparagine residue. In certain
embodiments wherein the first 51 N-terminal amino acids of the
protein are replaced by amino acids 1-11 from the protein ChR2, the
aspartate amino acid residue at position 156 (which corresponds to
original position 196 of the ReaChR amino acid sequence set forth
in SEQ ID NO: 14) is replaced by an asparagine residue.
[0203] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide, ibReaChR, comprises an amino acid
sequence having at least 58%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to the amino acid sequence set forth in SEQ ID NO: 53; and
includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123,
K117, R134, N246, K242, and Q258; and includes T128, S128 or A128,
where the amino acid numbering is as set forth in SEQ ID NO: 53. In
some embodiments, a subject hyperpolarizing light-responsive
polypeptide comprises an amino acid sequence having at least 58%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or 100%, amino acid sequence identity to the amino acid
sequence set forth in SEQ ID NO: 53; and includes S59, S90, S101,
S123, K117, R134, N246, K242, and Q258; and includes T128, where
the amino acid numbering is as set forth in SEQ ID NO: 53. In any
one of these embodiments, a subject anion channel polypeptide
comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV
(SEQ ID NO: 56)). In any one of these embodiments, a subject anion
channel polypeptide comprises an ER export signal (e.g., FCYENEV
(SEQ ID NO: 61)). In any one of these embodiments, a subject anion
channel polypeptide comprises both a membrane trafficking signal
(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export
signal (e.g., FCYENEV (SEQ ID NO: 61)).
[0204] In some embodiments, a subject hyperpolarizing
light-responsive polypeptide, ibReaChR, includes an amino acid
sequence having at least 58%, at least 60%, at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 98%, at least 99%, or 100%, amino acid sequence
identity to the amino acid sequence set forth in SEQ ID NO: 53; and
includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 of: S59, S90, S101, S123,
K117, R134, N246, K242, and Q258; includes T128, S128 or A128; and
includes A156 or N156, where the amino acid numbering is as set
forth in SEQ ID NO: 53. In some embodiments, a subject
hyperpolarizing light-responsive polypeptide comprises an amino
acid sequence having at least 58%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 98%, at least 99%, or 100%, amino acid
sequence identity to the amino acid sequence set forth in SEQ ID
NO: 53; and includes S59, S90, S101, S123, K117, R134, N246, K242,
and Q258; includes T128, S128 or A128; and includes A156 or N156,
where the amino acid numbering is as set forth in SEQ ID NO: 53. In
any one of these embodiments, a subject anion channel polypeptide
comprises a membrane trafficking signal (e.g., KSRITSEGEYIPLDQIDINV
(SEQ ID NO: 56)). In any one of these embodiments, a subject anion
channel polypeptide includes an ER export signal (e.g., FCYENEV
(SEQ ID NO: 61)). In any one of these embodiments, a subject anion
channel polypeptide comprises both a membrane trafficking signal
(e.g., KSRITSEGEYIPLDQIDINV (SEQ ID NO: 56)) and an ER export
signal (e.g., FCYENEV (SEQ ID NO: 61)).
Expression Vector
[0205] As noted above, aspects of the present disclosure include a
recombinant expression vector comprising a nucleic acid that
includes a D2SP. Suitable expression vectors include vectors
comprising a nucleotide sequence that encodes an RNA (e.g., an
mRNA) that when transcribed from the polynucleotides of the vector
will result in the expression of a subject gene product. In some
cases, the gene product is a polypeptide. In some cases, the gene
product encoded in the expression vector is a light-responsive
polypeptide that is expressed on the plasma membranes of the target
cells. In other instances, the gene product encoded in the
expression vector is a fluorescent protein that is expressed in the
cytosol of the target cells. Vectors which may be used include,
without limitation, lentiviral, herpes simplex virus, adenoviral,
and adeno-associated virus (AAV) vectors. Lentiviral vectors
include, but are not limited to human immunodeficiency virus
(HIV)-based vectors. Lentiviral vectors may be pseudotyped with the
envelope proteins of other viruses, including, but not limited to
vesicular stomatitis virus (VSV), rabies, Mo-murine leukemia virus
(MLV), baculovirus and Ebola. Such vectors may be prepared using
standard methods in the art.
[0206] Other vectors of interest include plasmid vectors. The term
plasmid as used herein can refer to nucleic acid, e.g., DNA derived
from a plasmid vector, cosmid, phagemid or bacteriophage, into
which one or more fragments of nucleic acid may be inserted or
cloned which encode for particular genes. This includes the
construction comprised of extrachromosomal genetic material,
usually of a circular duplex of DNA which can replicate
independently of chromosomal DNA in a host cell.
[0207] In certain embodiments, the recombinant expression vector
comprises multiple cloning sites that facilitate subcloning a
nucleotide sequence encoding a gene product of interest into the
recombinant expression vector, thereby operably linking the
nucleotide sequence encoding the gene product of interest to the
D2SP.
[0208] In some embodiments, a vector may be a recombinant AAV
vector. AAV vectors are DNA viruses of relatively small size that
can integrate, in a stable and site-specific manner, into the
genome of the cells that they infect. They are able to infect a
wide spectrum of cells without inducing any effects on cellular
growth, morphology or differentiation, and they do not appear to be
involved in human pathologies. The AAV genome has been cloned,
sequenced and characterized. It encompasses approximately 4700
bases and comprises an inverted terminal repeat (ITR) region of
approximately 145 bases at each end, which serves as an origin of
replication for the virus. The remainder of the genome is divided
into two essential regions that carry the encapsidation functions:
the left-hand part of the genome that comprises the rep gene
involved in viral replication and expression of the viral genes;
and the right-hand part of the genome that contains the cap gene
encoding the capsid proteins of the virus.
[0209] AAV vectors may be prepared using standard methods in the
art. Adeno-associated viruses of any serotype are suitable (see,
e.g., Blacklow, pp. 165-174 of "Parvoviruses and Human Disease" J.
R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P.
Tattersall "The Evolution of Parvovirus Taxonomy" In Parvoviruses
(J R Kerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.)
p5-14, Hudder Arnold, London, U K (2006); and D E Bowles, J E
Rabinowitz, R J Samulski "The Genus Dependovirus" (J R Kerr, S F
Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p15-23, Hudder
Arnold, London, UK (2006), the disclosures of each of which are
hereby incorporated by reference herein in their entireties).
Methods for purifying for vectors may be found in, for example,
U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 and
WO/1999/011764 titled "Methods for Generating High Titer
Helper-free Preparation of Recombinant AAV Vectors", the
disclosures of which are herein incorporated by reference in their
entirety. Methods of preparing AAV vectors in a baculovirus system
are described in, e.g., WO 2008/024998. AAV vectors can be
self-complementary or single-stranded. Preparation of hybrid
vectors is described in, for example, PCT Application No.
PCT/US2005/027091, the disclosure of which is herein incorporated
by reference in its entirety. The use of vectors derived from the
AAVs for transferring genes in vitro and in vivo has been described
(See e.g., International Patent Application Publication Nos.:
91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and
5,139,941; and European Patent No.: 0488528, all of which are
hereby incorporated by reference herein in their entireties). These
publications describe various AAV-derived constructs in which the
rep and/or cap genes are deleted and replaced by a gene of
interest, and the use of these constructs for transferring the gene
of interest in vitro (into cultured cells) or in vivo (directly
into an organism). The replication-defective recombinant AAVs
according to the present disclosure can be prepared by
co-transfecting a plasmid comprising the nucleic acid sequence of
interest flanked by two AAV inverted terminal repeat (ITR) regions,
and a plasmid carrying the AAV encapsidation genes (rep and cap
genes), into a cell line that is infected with a human helper virus
(for example an adenovirus). The AAV recombinants that are produced
are then purified by standard techniques.
[0210] In some embodiments, the vector(s) for use in the methods of
the present disclosure are encapsidated into a virus particle (e.g.
AAV virus particle including, but not limited to, AAV1, AAV2, AAV3,
AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13,
AAV14, AAV15, and AAV16). Accordingly, the present disclosure
includes a recombinant virus particle (recombinant because it
contains a recombinant polynucleotide) comprising any of the
vectors described herein. Methods of producing such particles are
known in the art and are described in U.S. Pat. No. 6,596,535, the
disclosure of which is hereby incorporated by reference in its
entirety. In some cases, AAV6 is used. In some cases, AAV1 is
used.
[0211] In some embodiments, the subject D2SP can be operably linked
to nucleotide sequences encoding various light-responsive
polypeptides (LRP), fluorescent proteins (XFP) and genetically
encoded indicators (GEI) for targeting D2 receptor-expressing
neuronal populations in mammalian brains. For example, the
following adeno associated vectors (AAVs) and components thereof
may be used without limitation: AAV-D2SP-LRP-XFP, AAV-D2SP-GEI,
AAV-D2SP-FLEX-LRP-XFP, AAV-D2SP-FLEX-GEI. Other AAV vectors that
may be used in association with the polynucleotides include those
with double floxed inverted reading frames (DIO) which allow
expression of proteins under the control of recombinases such as as
Cre and Flp: AAV-D2SP-DIO(Cre)-LRP-XFP (Cre-dependent expression),
AAV-D2SP-DIO(Flp)-LRP-XFP (Flp-dependent expression),
AAV-D2SP-DIO(Cre)-DIO(Flp)-LRP-XFP (Cre and Flp dependent
expression).
Genetically Modified Host Cell
[0212] The present disclosure provides isolated genetically
modified host cells (e.g., in vitro cells) that are genetically
modified with a subject nucleic acid. In some embodiments, a
subject isolated genetically modified host cell can produce a gene
product encoded by a nucleotide sequence operably linked to a D2SP
of the present disclosure.
[0213] Suitable host cells include eukaryotic host cells, such as a
mammalian cell. Mammalian cells of interest include human cells,
rodent cells, such as rat cells and mouse cells. Introduction of a
subject nucleic acid into the host cell can be effected, for
example by calcium phosphate precipitation, DEAE dextran mediated
transfection, liposome-mediated transfection, electroporation,
viral infection, or other known method.
[0214] Suitable mammalian cells include primary cells and
progenitor cells, such as stem cells. In some cases, the mammalian
cell is a neuron, e.g., a non-immortalized (primary) neuron. In
some embodiments, the cell is a neuronal cell or a neuronal-like
cell. The cells can be of human, non-human primate, mouse, or rat
origin, or derived from a mammal other than a human, non-human
primate, rat, or mouse. Suitable cell lines include, but are not
limited to, a human glioma cell line, e.g., SVGp12 (ATCC CRL-8621),
CCF-STTG1 (ATCC CRL-1718), SW 1088 (ATCC HTB-12), SW 1783 (ATCC
HTB-13), LLN-18 (ATCC CRL-2610), LNZTA3WT4 (ATCC CRL-11543),
LNZTA3WT11 (ATCC CRL-11544), U-138 MG (ATCC HTB-16), U-87 MG (ATCC
HTB-14), H4 (ATCC HTB-148), and LN-229 (ATCC CRL-2611); a human
medulloblastoma-derived cell line, e.g., D342 Med (ATCC HTB-187),
Daoy (ATCC HTB-186), D283 Med (ATCC HTB-185); a human tumor-derived
neuronal-like cell, e.g., PFSK-1 (ATCC CRL-2060), SK-N-DZ
(ATCCCRL-2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCC CRL-2142),
IMR-32 (ATCC CCL-127), etc.; a mouse neuronal cell line, e.g.,
BC3H1 (ATCC CRL-1443), EOC1 (ATCC CRL-2467), C8-D30 (ATCC
CRL-2534), C8-S(ATCC CRL-2535), Neuro-2a (ATCC CCL-131), NB41A3
(ATCC CCL-147), SW10 (ATCC CRL-2766), NG108-15 (ATCC HB-12317); a
rat neuronal cell line, e.g., PC-12 (ATCC CRL-1721), CTX TNA2 (ATCC
CRL-2006), C6 (ATCC CCL-107), F98 (ATCC CRL-2397), RG2 (ATCC
CRL-2433), B35 (ATCC CRL-2754), R3 (ATCC CRL-2764), SCP (ATCC
CRL-1700), OA1 (ATCC CRL-6538).
[0215] In some instances, the host cell is a progenitor cell or a
stem cell. "Stem cell," as used herein, refers to a cell having,
upon being induced, both the ability to differentiate into multiple
lineages of cells (multipotency or pluripotency) and the ability to
maintain its multipotency or pluripotency after cell division
(ability to self-renew). Stem cells encompass, for example,
hematopoietic stem cells, neural stem cells, hepatic stem cells,
dermal stem cells, germ stem cells, and embryonic stem (ES) or
induced pluripotent stem (iPS) cells, and stem cells induced from
these cells, etc. Stem cells can be obtained from embryonic,
post-natal, juvenile or adult tissue. The "progenitor cell" refers
to an undifferentiated cell derived from a stem cell, and is not
itself a stem cell. Some progenitor cells can produce progeny that
are capable of differentiating into more than one cell type.
[0216] In some embodiments, the host cell is a human ES cell. In
certain embodiments, the human ES cell can be differentiated into a
neuron. Any suitable method for growing and inducing
differentiation of ES cells may be used, some of which are
described in, e.g., U.S. Pat. Nos. 8,460,931 and 7,892,835; US App.
Pub. No. 20130252335 and 20100075416; PCT App. Pub. No.
WO2001/088100; and Kawasaki et al. Neuron 2000 28:31, which are
incorporated herein by reference. In other embodiments, the host
cell is an iPS cell, which are described in further detail in,
e.g., PCT App. Pub. No. WO2007/069666, which is incorporated herein
by reference.
Methods
[0217] As summarized above, aspects of the present disclosure
include a method of introducing into a target cell a nucleic acid
that includes a D2SP operably linked to a gene product that, when
expressed, performs a function of interest, e.g., light-induced
depolarization/hyperpolarization and/or fluorescent labeling.
Introducing the nucleic acid into a target cell may be done by any
convenient method, as described above for a genetically modified
host cell. The target cell may be in in vitro culture, or may be
located in vivo, e.g., a cell in a tissue in vivo, such as a
neuronal cell in the brain.
[0218] In certain embodiments, the target cell is a progenitor
cell, such as a neural progenitor cell. In some instances, the
target cell is a stem cell. Any convenient method for introducing a
nucleic acid into a progenitor cell or stem cell may be used to
introduce a nucleic acid that includes a D2SP operably linked to a
gene product, as described above with respect to a genetically
modified host cell.
[0219] In some embodiments, a target neuron is, e.g., a sensory
neuron, a motor neuron, or an interneuron. Target neurons of the
disclosure may include neurons of the central nervous system and/or
cells of the peripheral nervous system. In some embodiments, a
target tissue may include a plurality of nerve fibers, a nerve, a
nerve cell ganglion, a neuromuscular junction, a tissue that is
innervated by nerves, including but not limited to muscle, skin, or
endocrine tissue, or an anatomical region, such as a portion or
sub-portion of the brain or spinal cord. In some embodiments, a
target tissue may be a portion of an individual cell, such as a
specific axon of a nerve cell.
[0220] Exemplary target cells, brain regions and tissues include
but not limited to: basal ganglia, nucleus accumbens, cortex,
habenula, ventral tegmental area, substantia nigra, olfactory
tubercle, septum, amygdala, hippocampus, cerebellum, thalamus,
chemoreceptor trigger zone, pituitary gland, hypothalamus,
sympathetic ganglia, adrenal glands, peripheral afferent nerves,
enteric nerves, gastrointestinal mucosa, heart, pulmonary tissues,
vascular tissue, renal cortex and inner medulla of the kidney, and
glioblastomas.
[0221] A nucleic acid comprising a nucleotide sequence encoding a
gene product operably linked to a D2SP can be introduced into a
neuron by any convenient means. For example, a nucleic acid
comprising a nucleotide sequence encoding a gene product operably
linked to a D2SP can be introduced (e.g., injected) into a nerve
bundle or nerve fiber, such that the nucleic acid enters a neuron,
where the gene product operably linked to a D2SP is produced in the
neuron. A nucleic acid comprising a nucleotide sequence encoding a
gene product operably linked to a D2SP can be introduced (e.g.,
injected) proximal to a nerve. Stereotactic injection can be used;
see, e.g., Stein et al., J. Virol, 73:34243429, 1999; Davidson et
al., PNAS, 97:3428-3432, 2000; Davidson et al., Nat. Genet.
3:219-223, 1993; and Alisky & Davidson, Hum. Gene Ther.
11:2315-2329, 2000, the contents of each of which are hereby
incorporated by reference herein in their entireties.
[0222] Once the subject polynucleotides have been delivered to a
target neuron or tissue, the polynucleotides enter the target cells
and are expressed. In some embodiments, expression from the subject
nucleic acids only occurs in target cells wherein the D2SP is
active. In this way, if a subject polynucleotide is delivered to
cells other than a target cell, the polynucleotide will not be
expressed in the non-target cells because the D2SP will be inactive
in those cells. In some instances, the D2SP drives expression of a
gene product operably linked thereto with a high specificity.
Specificity of a promoter can be expressed as the number of cells
expressing a gene product operably linked to the promoter and
staining positively with an antibody specific to the D2 receptor
(such as Millipore ab1558; FIG. 3), divided by the total number of
cells expressing the gene product operably linked to the promoter.
In some instances, the D2SP drives expression of a gene product
operably linked thereto with a specificity of 91% or more, e.g.,
92% or more, 93% or more, 94% or more, 95% or more, 95.5% or more,
96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or
more, 98.1% or more, 98.2% or more, 98.3% or more, 98.4% or more,
or 98.5% or more. In some instances, the D2SP drives expression of
a gene product operably linked thereto with a specificity of 99% or
less, e.g., 99.5% or less, 99.3% or less, 99.1% or less, 99.0% or
less, 98.9% or less, 98.8% or less, 98.7% or less, 98.6% or less,
98.5% or less, 98.4% or less, 98.3% or less, or 98.2%. In some
instances, the D2SP drives expression of a gene product operably
linked thereto with a specificity in the range of 91 to 99%, e.g.,
92 to 99%, including 93 to 99%, 94 to 98.5%, or 95 to 98.5%. In
some instances, the D2SP drives expression of a gene product
operably linked thereto with a specificity of about 98.2%.
[0223] In some instances, the D2SP drives expression of a gene
product operably linked thereto with a percentage specificity that
is higher than the percentage specificity of expression of the gene
product driven by a conventional D2 receptor promoter, e.g. a D2
receptor promoter that includes exon 1 of the D2 receptor gene,
such as a nucleic acid having a sequence at least 90%, e.g., at
least 95%, at least 98%, at least 99% or 100% identical to the
sequence shown in SEQ ID NO: 2 (FIG. 2), by 5% or more, e.g., 6% or
more, 7% or more, 8% or more, including 9% or more. In some
instances, the D2SP drives expression of a gene product operably
linked thereto with a percentage specificity that is higher than
the percentage specificity of expression of the gene product driven
by a conventional D2 receptor promoter, e.g. a D2 receptor promoter
that includes exon 1 of the D2 receptor gene, such as a nucleic
acid having a sequence at least 90%, e.g., at least 95%, at least
98%, at least 99% or 100% identical to the sequence shown in SEQ ID
NO: 2 (FIG. 2), by 9% or less, e.g., 9.5% or less, 9.0% or less,
8.5% or less, including 8% or less. In some instances, the D2SP
drives expression of a gene product operably linked thereto with a
percentage specificity that is higher than the percentage
specificity of expression of the gene product driven by a
conventional D2 receptor promoter, e.g. a D2 receptor promoter that
includes exon 1 of the D2 receptor gene, such as a nucleic acid
having a sequence at least 90%, e.g., at least 95%, at least 98%,
at least 99% or 100% identical to the sequence shown in SEQ ID NO:
2 (FIG. 2), in the range of 5 to 9%, e.g., 6 to 9.5%, 6.5 to 9.0%,
7 to 8.5%, including 7.5 to 8.0%.
[0224] In some instances, the D2SP drives expression of a gene
product operably linked thereto with a high penetrance. Penetrance
of a promoter can be expressed as the number of cells expressing a
gene product operably linked to the promoter and staining
positively with an antibody specific to the D2 receptor (such as
Millipore ab1558; FIG. 3), divided by the total number of cells
staining positively with an antibody specific to the D2 receptor
(such as Millipore ab1558; FIG. 3). In some instances, the D2SP
drives expression of a gene product operably linked thereto with a
penetrance of 70% or more, e.g., 72% or more, 74% or more, 76% or
more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or
more, 83% or more, 84% or more, 85% or more, 86% or more, 86.5% or
more, 86.8% or more, or 87% or more. In some instances, the D2SP
drives expression of a gene product operably linked thereto with a
penetrance of 99% or less, e.g., 95% or less, 94% or less, 93% or
less, 92% or less, 91% or less, 90% or less, 89.5% or less, 89% or
less, 89.5% or less, 89% or less, 88.5% or less, 88% or less, 87.5%
or less, or 87% or less. In some instances, the D2SP drives
expression of a gene product operably linked thereto with a
penetrance in the range of 70 to 95%, e.g., 75 to 95%, including 78
to 93%, 79 to 91%, 80 to 90%, 81 to 89%, or 82 to 87%. In some
instances, the D2SP drives expression of a gene product operably
linked thereto with a penetrance of about 86.8%.
[0225] In some instances, the D2SP drives expression of a gene
product operably linked thereto with a percentage penetrance that
is higher than the percentage penetrance of expression of the gene
product driven by a conventional D2 receptor promoter, e.g. a D2
receptor promoter that includes exon 1 of the D2 receptor gene,
such as a nucleic acid having a sequence at least 90%, e.g., at
least 95%, at least 98%, at least 99% or 100% identical to the
sequence shown in SEQ ID NO: 2, by 5% or more, e.g., 8% or more,
10% or more, 12% or more, 14% or more, 16% or more, 17% or more,
including 18% or more. In some instances, the D2SP drives
expression of a gene product operably linked thereto with a
percentage penetrance that is higher than the percentage penetrance
of expression of the gene product driven by a conventional D2
receptor promoter, e.g. a D2 receptor promoter that includes exon 1
of the D2 receptor gene, such as a nucleic acid having a sequence
at least 90%, e.g., at least 95%, at least 98%, at least 99% or
100% identical to the sequence shown in SEQ ID NO: 2, by 35% or
less, e.g., 30% or less, 25% or less, 24% or less, 23% or less, 22%
or less, 21% or less, 20% or less, 19% or less, including 18% or
less. In some instances, the D2SP drives expression of a gene
product operably linked thereto with a percentage penetrance that
is higher than the percentage penetrance of expression of the gene
product driven by a conventional D2 receptor promoter, e.g. a D2
receptor promoter that includes exon 1 of the D2 receptor gene,
such as a nucleic acid having a sequence at least 90%, e.g., at
least 95%, at least 98%, at least 99% or 100% identical to the
sequence shown in SEQ ID NO: 2, in the range of 5 to 35%, e.g., 8
to 30%, 10 to 25%, 12 to 20%, 14 to 19%, including 16 to 18%.
[0226] In certain instances, the gene product operably linked to a
D2SP in a nucleic acid introduced into a target cell is a
light-responsive polypeptide, as described above and elsewhere
herein. When the gene product is a light-responsive polypeptide
operably linked to a D2SP, the light-responsive polypeptide
expressed in the target cell, such as a target neuron, can modulate
the activity of the target cell by inducing hyperpolarization or
depolarization of the target cell when the polypeptide is activated
by light. In some instances, the activity modulated by activation
of the light-responsive polypeptide is the pattern or amplitude of
action potential firing, the resting potential, subthreshold
changes in membrane potential, activity-dependent transcription
and/or translation of a gene, and the like, in a target neuron.
[0227] In some embodiments, a light-activated polypeptide, when
expressed on the membrane of a cell (e.g., a mammalian cell), and
when exposed to light of an activating wavelength, hyperpolarizes
the membrane. In some embodiments, a light-activated polypeptide
exhibits prolonged stability of photocurrents. In some embodiments,
a light-activated polypeptide exhibits enhanced expression in cell
membranes and larger photocurrents in cultured neurons. In some
embodiments, a subject light-activated polypeptide exhibits
decelerated channel kinetics/decelerated channel closure. In some
embodiments, a light-activated polypeptide conduct anions and
inhibits the formation of action potentials in neurons for an
extended period of time (e.g., from about 0.5 hours, up to about
0.75 hours, up to about 1 hour, up to about 1.25 hours, up to about
1.5 hours, up to about 1.75 hours, up to about 2 hours, up to about
2.25 hours, up to about 2.5 hours, up to about 2.75 hours, up to
about 3 hours or more) after brief light stimulations at lower
light intensities.
[0228] In some instances, the gene product operably linked to a
D2SP in a nucleic acid introduced into a target cell is a
fluorescent protein polypeptide, as described above and elsewhere
herein. When the gene product is a fluorescent protein operably
linked to a D2SP, the fluorescent protein expressed in the target
cell, such as a target neuron, can fluorescently label the target
cell by emitting light when the protein is stimulated by light of
an appropriate wavelength, as described above. In certain
embodiments, the fluorescent protein is a genetically encoded
indicator, such as a calcium indicator or a voltage indicator. When
the gene product is a genetically encoded indicator operably linked
to a D2SP, the genetically encoded indicator expressed in the
target cell, such as a target neuron, alters its fluorescence
properties, such as intensity, excitation and/or emission
wavelengths, etc.
[0229] Any convenient means may be used to deliver light to the
target cell or neuron expressing a gene product operably linked to
a D2SP, thereby modulating or fluorescently labeling the target
cell. A target cell in culture or in an ex vivo tissue slice may be
subjected to light using a fluorescent microscope, a target cell in
suspension may be subjected to light using fluorescence activated
cell sorting (FACS) device or a fluorimeter, and so on.
[0230] In some cases, the light is delivered transdermally or
transcutaneously to a target cell or neuron in vivo. In some cases,
an implantable light source is used; and the light is delivered to
a site within the body. In some cases, the light is delivered to a
treatment site within the body. In some cases, the light is
delivered intracranially.
[0231] In some cases, the light used to activate a light-responsive
polypeptide expressed in a neuron has an intensity of from about
0.05 mW/mm.sup.2 to about 0.1 mW/mm.sup.2, from about 0.1
mW/mm.sup.2 to about 0.2 mW/mm.sup.2, from about 0.2 mW/mm.sup.2 to
about 0.3 mW/mm.sup.2, from about 0.3 mW/mm.sup.2 to about 0.4
mW/mm.sup.2, from about 0.4 mW/mm.sup.2 to about 0.5 mW/mm.sup.2,
from about 0.5 mW/mm.sup.2 to about 0.6 mW/mm.sup.2, from about 0.6
mW/mm.sup.2 to about 0.7 mW/mm.sup.2, from about about 0.7
mW/mm.sup.2 to about 0.8 mW/mm.sup.2, from about 0.8 mW/mm.sup.2 to
about 0.9 mW/mm.sup.2, or from about about 0.9 mW/mm.sup.2 to about
1.0 mW/mm.sup.2. In some cases, the light used to activate a
light-responsive polypeptide expressed in a neuron has an intensity
of from about 1.0 mW/mm.sup.2 to about 1.1 mW/mm.sup.2, from about
1.1 mW/mm.sup.2 to about 1.2 mW/mm.sup.2, from about 1.2
mW/mm.sup.2 to about 1.3 mW/mm.sup.2, from 1.3 mW/mm.sup.2 to about
1.4 mW/mm.sup.2, from about 1.4 mW/mm.sup.2 to about 1.5
mW/mm.sup.2, from about 1.5 mW/mm.sup.2 to about 1.6 mW/mm.sup.2,
from about 1.6 mW/mm.sup.2 to about 1.7 mW/mm.sup.2, from about 1.7
mW/mm.sup.2 to about 1.8 mW/mm.sup.2, from about 1.8 mW/mm.sup.2 to
about 1.9 mW/mm.sup.2, from about 1.9 mW/mm.sup.2 to about 2.0
mW/mm.sup.2, from about 2.0 mW/mm.sup.2 to about 2.5 mW/mm.sup.2,
from about 2.5 mW/mm.sup.2 to about 3 mW/mm.sup.2, from about 3
mW/mm.sup.2 to about 3.5 mW/mm.sup.2, from about 3.5 mW/mm.sup.2 to
about 4 mW/mm.sup.2, from about 4 mW/mm.sup.2 to about 4.5
mW/mm.sup.2, from about 4.5 mW/mm.sup.2 to about 5 mW/mm.sup.2,
from about 5 mW/mm.sup.2 to about 5.5 mW/mm.sup.2, from about 5.5
mW/mm.sup.2 to about 6 mW/mm.sup.2, from about 6 mW/mm.sup.2 to
about 7 mW/mm.sup.2, or from about 7 mW/mm.sup.2 to about 10
mW/mm.sup.2. In some cases, the light used to activate a
light-responsive polypeptide expressed in a neuron has an intensity
of from about 0.05 mW/mm.sup.2 to about 0.1 mW/mm.sup.2. In some
cases, the light used to activate a light-responsive polypeptide
expressed in a neuron has an intensity of about 0.25 mW/mm.sup.2.
In some cases, the light used to activate a light-responsive
polypeptide expressed in a neuron has an intensity of about 1
mW/mm.sup.2.
Utility
[0232] The subject nucleic acids, genetically modified host cells
and methods find use in a wider variety of applications, including
transfecting, identifying, targeting, and isolating live
D2R-expressing cells derived from healthy or afflicted human and
animal subject populations, as well as transfection,
identification, and isolation of D2R-expressing cells from
stem/progenitor-cell populations from healthy or afflicted
subjects, for in-vitro/ex-vivo genetic, proteomic, transcriptomic,
electrophysiological, and pharmacologic analyses.
[0233] A nucleic acid comprising a D2SP may find use in enrichment
of D2R-expressing cells through cell-sorting techniques such as
fluorescent-activated cell sorting (FACS), not only for analysis
and characterization of the cell population associated with dozens
of dopamine-related disorders, but also for the purpose of
therapeutic transplantation of the D2R-expressing cells.
[0234] In some embodiments, factors that participate in induction
of cells to differentiate into dopaminoceptive neurons may be
identified using a D2SP to study D2R-expressing cultured cells and
D2R-expressing human-derived stem cells as well as nonhuman-derived
stem cells. In certain embodiments, graft cells for drug addiction,
obesity, gambling disorder and others may be obtained from
undifferentiated cells using a D2SP to identify the relevant cell
populations for grafting. In other cases, novel drugs for treatment
may be developed based on the dopaminoceptive neurons'
differentiating and inducing factors identified using cells
identified based on D2SP-driven expression of a fluorescent
protein. The subject nucleic acid and method of using the same
enable targeting of virally-mediated optogenetic constructs, RNA or
DNA-based therapies, and other gene-therapy approaches in patient
populations, both in isolation and in combination with
pharmacologic, direct-stimulation, or antibody-based
interventions.
[0235] In some embodiments, the subject nucleic acid and method may
be used to target expression of gene products for the study and
treatment of both central and peripheral disorders, which include
but are not limited to: schizophrenia, gambling disorder, drug
addiction, Tourette's syndrome, multiple system atrophy,
supranuclear palsy, parkinson's disease, dementia, autism, ADHD,
depression, tardive dyskinesia, glioblastoma, compulsive/impulsive
sexual behavior, compulsive spending, obesity, functional
dyspepsia, gastric stasis, emesis, diabetic gastroparesis,
irritable bowel syndrome, Cushing's disease, hypertension, and
renal inflammation/injury, and hyperprolactinaemia with associated
alterations, such as gynaecomastia, galactorrhoea, amenorrhoea and
impotence. D2R-expressing cells may also be characterized to
provide animal models of these diseases, on which more detailed
characterization and drug/therapeutic screening can be
performed.
Kits
[0236] Further aspects of the present disclosure include a kit that
includes a recombinant expression vector, as described above,
comprising the subject nucleic acid, i.e., a nucleic acid
comprising a dopamine receptor type 2-specific promoter (D2SP),
wherein the D2SP does not include exon 1 of a D2 receptor gene,
wherein the D2SP includes a Kozak sequence, and wherein the D2SP
comprises a nucleotide sequence having at least 95% sequence
identity to the nucleotide sequence set forth in SEQ ID NO: 1. In
certain embodiments, the recombinant expression vector of the
subject kit comprises multiple cloning sites, or equivalents
thereof, that facilitate subcloning a nucleotide sequence encoding
a gene product of interest to a user into the recombinant
expression vector, thereby operably linking the nucleotide sequence
encoding the gene product of interest to the D2SP.
[0237] In certain embodiments, the recombinant expression vector of
the subject kit comprises a nucleotide sequence encoding a
light-responsive polypeptide, a genetically encoded indicator
and/or a fluorescent protein operably linked to the D2SP.
[0238] The kit may also include a control expression vector, such
as a positive control expression vector and/or a negative control
expression vector. In some embodiments, the positive control
expression vector comprises a nucleic acid encoding a known gene
product, such as a light-responsive polypeptide or a fluorescent
polypeptide as described above, operably linked to the D2SP. In
some instances, the positive control expression vector contains a
nucleic acid encoding a fluorescent protein, such as a green
fluorescent protein, a yellow fluorescent protein, or a red
fluorescent protein.
[0239] Components of a subject kit can be in separate containers;
or can be combined in a single container.
[0240] In addition to above-mentioned components, a subject kit can
further include instructions for using the components of the kit
and to practice the subject methods. The instructions for
practicing the subject methods are generally recorded on a suitable
recording medium. For example, the instructions may be printed on a
substrate, such as paper or plastic, etc. As such, the instructions
may be present in the kits as a package insert, in the labeling of
the container of the kit or components thereof (i.e., associated
with the packaging or subpackaging) etc. In other embodiments, the
instructions are present as an electronic storage data file present
on a suitable computer readable storage medium, e.g. CD-ROM,
diskette, flash drive, etc. In yet other embodiments, the actual
instructions are not present in the kit, but means for obtaining
the instructions from a remote source, e.g. via the internet, are
provided. An example of this embodiment is a kit that includes a
web address where the instructions can be viewed and/or from which
the instructions can be downloaded. As with the instructions, this
means for obtaining the instructions is recorded on a suitable
substrate.
Examples
[0241] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Celsius, and pressure
is at or near atmospheric. Standard abbreviations may be used,
e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or
sec, second(s); min, minute(s); h or hr, hour(s); aa, amino
acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s);
i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c.,
subcutaneous(ly); and the like.
Example 1: Protocol for Antibody Staining Cells Expressing a Type 2
Dopamine Receptor
[0242] The following protocols were used to stain cells in a fixed
tissue section with a type 2 dopamine (D2) receptor-specific
antibody.
[0243] A. Standard Staining Protocol.
[0244] 1) Rinsed 40 .mu.m sections in phosphate-buffered saline
(PBS) (pH 7.4) 3.times.10 minutes.
[0245] 2) Blocked in PBS+3% normal donkey serum+0.3% Triton-X for
30 minutes (PBS++)
[0246] 3) Incubated in primary antibody (rabbit anti-D2R, millipore
ab1558) 1:500 in PBS++ overnight at 4.degree. C. on a rotary
shaker.
[0247] 4) Washed slices 4.times.15 minutes in PBS
[0248] 5) Incubated in secondary antibody (Alexa-fluor 647, goat
anti-rabbit, Life Technologies A-21245) 1:500 in PBS++ for 3 hours
at room temperature.
[0249] 6) Washed for 15 min in PBS
[0250] 7) Washed for 15 min in 1:50000
4',6-diamidino-2-phenylindole (DAPI) in PBS
[0251] 8) Washed for 15 min in PBS
[0252] The above protocol produced the staining pattern seen in
FIG. 3A.
[0253] B. Modified Staining Protocol.
[0254] 1) Rinsed 40 .mu.m sections in PBS (pH 7.4) 3.times.10
minutes.
[0255] 2) Blocked in PBS+3% normal donkey serum+0.3% Triton-X for
30 minutes (PBS++)
[0256] 3) Incubated in primary antibody (rabbit anti-D2R, millipore
ab1558) 1:200 in PBS++24 hrs at room temperature on a rotary
shaker.
[0257] 4) Washed slices 4.times.15 minutes in PBS
[0258] 5) Incubated in secondary antibody (Alexa-fluor 647, goat
anti-rabbit, Life Technologies A-21245) 1:500 in PBS++ for 8 hours
at room temperature.
[0259] 6) Washed slices 4.times.15 minutes in PBS
[0260] 7) Incubated in tertiary antibody (Alexa-fluor 647, donkey
anti-goat, Life Technologies A-21447) 1:500 in PBS++ for 8 hours at
room temperature.
[0261] 8) Washed for 15 min in PBS
[0262] 9) Washed for 15 min in 1:50000 DAPI in PBS
[0263] 10) Washed for 15 min in PBS
[0264] The above protocol produced the staining pattern seen in
FIG. 3B.
Example 2: D2SP Drives Expression in Rat Hippocampal Primary
Neurons
[0265] Rat Hippocampal primary neurons were transfected with
D2SP-eNpHR 3.0-EYFP and stained for D2R using the modified staining
procedure described in Example 1 (FIG. 4). The green color is from
the EYFP, showing the cells expressing D2SP-eNpHR 3.0-EYFP and blue
shows all Dopamine Receptor 2 cells.
Example 3: Comparison of Expression of eNpHR 3.0-EYFP Under D2SP
and D2R
[0266] With reference to FIG. 5, the middle panels show EYFP,
showing the cells expressing D2SP-eNpHR 3.0-EYFP (top) or D2R-eNpHR
3.0-EYFP (bottom) and the left panels show all Dopamine Receptor 2
cells. The right panels for each promoter construct show the merge
of the two previous panels.
[0267] The specificity and penetrance of the two promoters were
also compared and are shown in Table 1.
TABLE-US-00001 TABLE 1 Specificity Penetrance D2SP::NY 112/114 =
98.2% 112/129 = 86.8% D2R::NY 76/84 = 90.5% 76/110 = 69%
Example 4: Recombinant Expression Vectors Containing D2SP
[0268] The following recombinant expression vectors that contain
D2SP operably linked to nucleotides sequences encoding one or more
gene products were constructed:
[0269] pAAV-D2SP-hChR2(H134R)-EYFP (FIG. 6);
[0270] pAAV-D2SP-ehChR2(H134R)-EYFP (FIG. 7);
[0271] pAAV-D2SP-eNpHR 3.0-EYFP (FIG. 8);
[0272] pAAV-D2SP-SwiChRca-TS-EYFP (FIG. 9);
[0273] pAAV-D2SP-EYFP (FIG. 10);
[0274] pAAV-D2SP-GCaMP 6f (FIG. 11);
[0275] pAAV-D2SP-GCaMP 6m (FIG. 12);
[0276] pAAV-D2SP-mCherry-IRES-Cre (FIG. 13); and
[0277] pAAV-D2SP-mCherry-IRES-Flpo (FIG. 14).
[0278] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
6611552DNAArtificial sequenceSynthetic nucleic acid 1ttatcctcgg
tgcatctcag agaaataagc attgcttgga ccaatgtgga ccggatgtta 60acacctagag
ccagagagat taaaaaattt aatcaacatc tacaactggc aagggataga
120cataggacac acgactgggt ggaaaacgta tagaggtgat gggttgagaa
gaacaaaatc 180cctgtttaag taggttattt cttgggaaga acatgtccag
ggcacatagg aaaatagtgg 240ggattcaacc atgtctgtaa tgtgtgagtg
ccttaaaagc aaatgtgaaa aattctaatg 300tttctggtag ttctaacact
tccctaccat gcctatagag agccatgaat agaccatacc 360ccaagaataa
tgaatagggg aaggggaggc tagttcccct tttcttaaat gcctccataa
420ctggccacat ctaagaaaaa tgtgctgtgt atagggactg ttccactgct
ggttccccgt 480gaggtttgga ggggcatgcc tctttgggtc ccagattcca
cctttgaaat caaacagggg 540ttagttgaat attagtgtct gtctttccaa
ccttaatttt ccaggattgt gtggatcaat 600ggaaggagtt tcttctttgt
ggctaagtgg catgactgcc ggctatatgc agactgtcct 660ctgtgctcct
gcccttggaa ttctgtggtg ccttctcctt ggggacttga attggccaat
720ggccagctcc tgtgaggtct ccggagctgt cggtactcca cagcacctat
ttaagctaca 780agtatttgga agactctact ctggattgac cccatgcatt
ctgaatctca tgtagaagct 840ggccaaggca ggacagaggg acagaaagca
ccagctggat ttgagaagaa gaggatggaa 900agggttgtag gttccctggg
tgggagatga ccctggacag ggctgaagaa gatcacattt 960ctcttcctcc
tgctcctcag tgcagacgga agggtgagct agaattttca cggccttctt
1020tatcattccc atcttagatc tgctctgccc aagtcttcct ctcagaaagc
acaacagcag 1080aacgaactgc tgtgattttc agacctgagg tctgtacacc
gactctggat atccttccgg 1140aatctatttc tcctttaaag acttgatgta
ccacacgtag tgcttcagct agcccttggc 1200cctgactcct caaaggaggg
gatcgacccg ctggtgttgt gattgctaga ccagagtagg 1260tttggatggg
cagggtgtta cttaaaaagt ataggatgac accggcgagc agtccggagc
1320acaggctatc cccactcaaa gccagagatg gattctcggt ctcagctctc
aaggttcctt 1380ccccaggccc cacagtgcag agatagttct ggggccctgg
gtgggtgggg cctctgtaca 1440aggggcgggg ttcccgggcg cctcgtggcc
agggtgaccc cgccccctcc tcctgcgcag 1500cgctctgatt ccgcggagct
gtccagcctc agtgccgggg ggatccgcca cc 155221810DNAArtificial
sequenceSynthetic nucleic acid 2ttatcctcgg tgcatctcag agaaataagc
attgcttgga ccaatgtgga ccggatgtta 60acacctagag ccagagagat taaaaaattt
aatcaacatc tacaactggc aagggataga 120cataggacac acgactgggt
ggaaaacgta tagaggtgat gggttgagaa gaacaaaatc 180cctgtttaag
taggttattt cttgggaaga acatgtccag ggcacatagg aaaatagtgg
240ggattcaacc atgtctgtaa tgtgtgagtg ccttaaaagc aaatgtgaaa
aattctaatg 300tttctggtag ttctaacact tccctaccat gcctatagag
agccatgaat agaccatacc 360ccaagaataa tgaatagggg aaggggaggc
tagttcccct tttcttaaat gcctccataa 420ctggccacat ctaagaaaaa
tgtgctgtgt atagggactg ttccactgct ggttccccgt 480gaggtttgga
ggggcatgcc tctttgggtc ccagattcca cctttgaaat caaacagggg
540ttagttgaat attagtgtct gtctttccaa ccttaatttt ccaggattgt
gtggatcaat 600ggaaggagtt tcttctttgt ggctaagtgg catgactgcc
ggctatatgc agactgtcct 660ctgtgctcct gcccttggaa ttctgtggtg
ccttctcctt ggggacttga attggccaat 720ggccagctcc tgtgaggtct
ccggagctgt cggtactcca cagcacctat ttaagctaca 780agtatttgga
agactctact ctggattgac cccatgcatt ctgaatctca tgtagaagct
840ggccaaggca ggacagaggg acagaaagca ccagctggat ttgagaagaa
gaggatggaa 900agggttgtag gttccctggg tgggagatga ccctggacag
ggctgaagaa gatcacattt 960ctcttcctcc tgctcctcag tgcagacgga
agggtgagct agaattttca cggccttctt 1020tatcattccc atcttagatc
tgctctgccc aagtcttcct ctcagaaagc acaacagcag 1080aacgaactgc
tgtgattttc agacctgagg tctgtacacc gactctggat atccttccgg
1140aatctatttc tcctttaaag acttgatgta ccacacgtag tgcttcagct
agcccttggc 1200cctgactcct caaaggaggg gatcgacccg ctggtgttgt
gattgctaga ccagagtagg 1260tttggatggg cagggtgtta cttaaaaagt
ataggatgac accggcgagc agtccggagc 1320acaggctatc cccactcaaa
gccagagatg gattctcggt ctcagctctc aaggttcctt 1380ccccaggccc
cacagtgcag agatagttct ggggccctgg gtgggtgggg cctctgtaca
1440aggggcgggg ttcccgggcg cctcgtggcc agggtgaccc cgccccctcc
tcctgcgcag 1500cgctctgatt ccgcggagct gtccagcctc agtgccgggg
ctggtcccct cttgtgcgcg 1560gcgcctcctg gccggcttcc cgcctggttc
ccgcgctggg ctcccgtcct cccgccccgc 1620cttcgtcctg ccccgccgcg
gccggtctac tgctccccgc gggcccgagc cggccgagcg 1680gctgcccgcc
ggggatctga acggcgcggc ggggccggaa gccgagggac ccgcggaggg
1740gaccggcggc cccggacggc tgccggaggg gcggccgtgc gtggatgcgg
cgggagctgg 1800aagcctcgag 18103313DNAArtificial sequenceSynthetic
nucleic acid 3ctggtcccct cttgtgcgcg gcgcctcctg gccggcttcc
cgcctggttc ccgcgctggg 60ctcccgtcct cccgccccgc cttcgtcctg ccccgccgcg
gccggtctac tgctccccgc 120gggcccgagc cggccgagcg gctgcccgcc
ggggatctga acggcgcggc ggggccggaa 180gccgagggac ccgcggaggg
gaccggcggc cccggacggc tgccggaggg gcggccgtgc 240gtggatgcgg
cgggagctgg aagcctcgag cagccggcgc cttctctggc cccgggcgcc
300atatggcttg aag 3134310PRTArtificial sequenceSynthetic
polypeptide 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 Pro305
3105340PRTArtificial sequenceSynthetic polypeptide 5Met 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 Pro Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu305 310 315
320Gly Glu Tyr Ile Pro Leu Asp Gln Ile Asp Ile Asn Val Phe Cys Tyr
325 330 335Glu Asn Glu Val 3406310PRTArtificial sequenceSynthetic
polypeptide 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 Ser 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 Ala 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 Pro305
3107340PRTArtificial sequenceSynthetic polypeptide 7Met 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 Ser 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 Ala 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 Pro Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu305 310 315
320Gly Glu Tyr Ile Pro Leu Asp Gln Ile Asp Ile Asn Val Phe Cys Tyr
325 330 335Glu Asn Glu Val 3408300PRTArtificial sequenceSynthetic
polypeptide 8Met 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
290 295 3009330PRTArtificial sequenceSynthetic polypeptide 9Met 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 Ala Ala Ala Lys 290 295
300Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile
Asp305 310 315 320Ile Asn Val Phe Cys Tyr Glu Asn Glu Val 325
33010344PRTArtificial sequenceSynthetic polypeptide 10Met Ser Arg
Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala
Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val
Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His Glu 35 40
45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val
50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu
Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile
Leu Gln Trp 85 90 95Ile Thr Phe Ala Leu Ser Ala Leu Cys Leu Met Phe
Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp Glu Glu
Ile Tyr Val Ala Thr Ile 115 120 125Glu Met Ile Lys Phe Ile Ile Glu
Tyr Phe His Glu Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser
Asn Gly Asn Lys Thr Val Trp Leu Arg Tyr145 150 155 160Ala Glu Trp
Leu Leu Thr Cys Pro Val Leu Leu Ile His Leu Ser Asn 165 170 175Leu
Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185
190Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys
195 200 205Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser
Tyr Gly 210 215 220Met Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile
Glu Ala Phe His225 230 235 240Thr Val Pro Lys Gly Ile Cys Arg Glu
Leu Val Arg Val Met Ala Trp 245 250 255Thr Phe Phe Val Ala Trp Gly
Met Phe Pro Val Leu Phe Leu Leu Gly 260 265 270Thr Glu Gly Phe Gly
His Ile Ser Pro Tyr Gly Ser Ala Ile Gly His 275 280 285Ser Ile Leu
Asp Leu Ile Ala Lys Asn Met Trp Gly Val Leu Gly Asn 290 295 300Tyr
Leu Arg Val Lys Ile His Glu His Ile Leu Leu Tyr Gly Asp Ile305 310
315 320Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val
Glu 325 330 335Thr Leu Val Ala Glu Glu Glu Asp
34011374PRTArtificial sequenceSynthetic polypeptide 11Met Ser Arg
Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala
Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val
Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His Glu 35 40
45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val
50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu
Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile
Leu Gln Trp 85 90 95Ile Thr Phe Ala Leu Ser Ala Leu Cys Leu Met Phe
Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp Glu Glu
Ile Tyr Val Ala Thr Ile 115 120 125Glu Met Ile Lys Phe Ile Ile Glu
Tyr Phe His Glu Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser
Asn Gly Asn Lys Thr Val Trp Leu Arg Tyr145 150 155 160Ala Glu Trp
Leu Leu Thr Cys Pro Val Leu Leu Ile His Leu Ser Asn 165 170 175Leu
Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185
190Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys
195 200 205Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser
Tyr Gly 210 215 220Met Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile
Glu Ala Phe His225 230 235 240Thr Val Pro Lys Gly Ile Cys Arg Glu
Leu Val Arg Val Met Ala Trp 245 250 255Thr Phe Phe Val Ala Trp Gly
Met Phe Pro Val Leu Phe Leu Leu Gly 260 265 270Thr Glu Gly Phe Gly
His Ile Ser Pro Tyr Gly Ser Ala Ile Gly His 275 280 285Ser Ile Leu
Asp Leu Ile Ala Lys Asn Met Trp Gly Val Leu Gly Asn 290 295 300Tyr
Leu Arg Val Lys Ile His Glu His Ile Leu Leu Tyr Gly Asp Ile305 310
315 320Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val
Glu 325 330 335Thr Leu Val Ala Glu Glu Glu Asp Ala Ala Ala Lys Ser
Arg Ile Thr 340 345 350Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile
Asp Ile Asn Val Phe 355 360 365Cys Tyr Glu Asn Glu Val
37012348PRTArtificial sequenceSynthetic polypeptide 12Met Ser Arg
Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala
Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val
Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His Glu 35 40
45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val
50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu
Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile
Leu Gln Trp 85 90 95Ile Thr Phe Ala Leu Ser Ala Leu Cys Leu Met Phe
Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp Glu Glu
Ile Tyr Val Ala Thr Ile 115 120 125Glu Met Ile Lys Phe Ile Ile Glu
Tyr Phe His Glu Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser
Asn Gly Asn Lys Thr Val Trp Leu Arg Tyr145 150 155 160Ala Glu Trp
Leu Leu Thr Cys Pro Val Ile Leu Ile His Leu Ser Asn 165 170 175Leu
Thr Gly Leu Ala Asn Asp Tyr Asn Lys Arg Thr Met Gly Leu Leu 180 185
190Val Ser Asp Ile Gly Thr Ile Val Trp Gly Thr Thr Ala Ala Leu Ser
195 200 205Lys Gly Tyr Val Arg Val Ile Phe Phe Leu Met Gly Leu Cys
Tyr Gly 210 215 220Ile Tyr Thr Phe Phe Asn Ala Ala Lys Val Tyr Ile
Glu Ala Tyr His225 230 235 240Thr Val Pro Lys Gly Arg Cys Arg Gln
Val Val Thr Gly Met Ala Trp 245 250 255Leu Phe Phe Val Ser Trp Gly
Met Phe Pro Ile Leu Phe Ile Leu Gly 260 265 270Pro Glu Gly Phe Gly
Val Leu Ser Val Tyr Gly Ser Thr Val Gly His 275 280 285Thr Ile Ile
Asp Leu Met Ser Lys Asn Cys Trp Gly Leu Leu Gly His 290 295 300Tyr
Leu Arg Val Leu Ile His Glu His Ile Leu Ile His Gly Asp Ile305 310
315 320Arg Lys Thr Thr Lys Leu Asn Ile Gly Gly Thr Glu Ile Glu Val
Glu 325 330 335Thr Leu Val Glu Asp Glu Ala Glu Ala Gly Ala Val 340
34513378PRTArtificial sequenceSynthetic polypeptide 13Met Ser Arg
Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala
Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val
Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His Glu 35 40
45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val
50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu
Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile
Leu Gln Trp 85 90 95Ile Thr Phe Ala Leu Ser Ala Leu Cys Leu Met Phe
Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp Glu Glu
Ile Tyr Val Ala Thr Ile 115 120 125Glu Met Ile Lys Phe Ile Ile Glu
Tyr Phe His Glu Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser
Asn Gly Asn Lys Thr Val Trp Leu Arg Tyr145 150 155 160Ala Glu Trp
Leu Leu Thr Cys Pro Val Ile Leu Ile His Leu Ser Asn 165 170 175Leu
Thr Gly Leu Ala Asn Asp Tyr Asn Lys Arg Thr Met Gly Leu Leu 180 185
190Val Ser Asp Ile Gly Thr Ile Val Trp Gly Thr Thr Ala Ala Leu Ser
195 200 205Lys Gly Tyr Val Arg Val Ile Phe Phe Leu Met Gly Leu Cys
Tyr Gly 210 215 220Ile Tyr Thr Phe Phe Asn Ala Ala Lys Val Tyr Ile
Glu Ala Tyr His225 230 235 240Thr Val Pro Lys Gly Arg Cys Arg Gln
Val Val Thr Gly Met Ala Trp 245 250 255Leu Phe Phe Val Ser Trp Gly
Met Phe Pro Ile Leu Phe Ile Leu Gly 260 265 270Pro Glu Gly Phe Gly
Val Leu Ser Val Tyr Gly Ser Thr Val Gly His 275 280 285Thr Ile Ile
Asp Leu Met Ser Lys Asn Cys Trp Gly Leu Leu Gly His 290 295 300Tyr
Leu Arg Val Leu Ile His Glu His Ile Leu Ile His Gly Asp Ile305 310
315 320Arg Lys Thr Thr Lys Leu Asn Ile Gly Gly Thr Glu Ile Glu Val
Glu 325 330 335Thr Leu Val Glu Asp Glu Ala Glu Ala Gly Ala Val Ala
Ala Ala Lys 340 345 350Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro
Leu Asp Gln Ile Asp 355 360 365Ile Asn Val Phe Cys Tyr Glu Asn Glu
Val 370 37514350PRTArtificial sequenceSynthetic polypeptide 14Met
Val Ser Arg Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala1 5 10
15Leu Ala Ala Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val
20 25 30Pro Val Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala
His 35 40 45Glu Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn
Gly Ser 50 55 60Val Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu
Ala Trp Leu65 70 75 80Lys Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala
Ala Asn Ile Leu Gln 85 90 95Trp Val Thr Phe Ala Leu Ser Val Ala Cys
Leu Gly Trp Tyr Ala Tyr 100 105 110Gln Ala Trp Arg Ala Thr Cys Gly
Trp Glu Glu Val Tyr Val Ala Leu 115 120 125Ile Glu Met Met Lys Ser
Ile Ile Glu Ala Phe His Glu Phe Asp Ser 130 135 140Pro Ala Thr Leu
Trp Leu Ser Ser Gly Asn Gly Val Val Trp Met Arg145 150 155 160Tyr
Gly Glu Trp Leu Leu Thr Cys Pro Val Ile Leu Ile His Leu Ser 165 170
175Asn Leu Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr Met Gly Leu
180 185 190Leu Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr Ser
Ala Met 195 200 205Cys Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile
Ser Leu Ser Tyr 210 215 220Gly Met Tyr Thr Tyr Phe His Ala Ala Lys
Val Tyr Ile Glu Ala Phe225 230 235 240His Thr Val Pro Lys Gly Leu
Cys Arg Gln Leu Val Arg Ala Met Ala 245 250 255Trp Leu Phe Phe Val
Ser Trp Gly Met Phe Pro Val Leu Phe Leu Leu 260 265 270Gly Pro Glu
Gly Phe Gly His Ile Ser Pro Tyr Gly Ser Ala Ile Gly 275 280 285His
Ser Ile Leu Asp Leu Ile Ala Lys Asn Met Trp Gly Val Leu Gly 290 295
300Asn Tyr Leu Arg Val Lys Ile His Glu His Ile Leu Leu Tyr Gly
Asp305 310 315 320Ile Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln
Glu Met Glu Val 325 330 335Glu Thr Leu Val Ala Glu Glu Glu Asp Lys
Tyr Glu Ser Ser 340 345 35015380PRTArtificial sequenceSynthetic
polypeptide 15Met Val Ser Arg Arg Pro Trp Leu Leu Ala Leu Ala Leu
Ala Val Ala1 5 10 15Leu Ala Ala Gly Ser Ala Gly Ala Ser Thr Gly Ser
Asp Ala Thr Val 20 25 30Pro Val Ala Thr Gln Asp Gly Pro Asp Tyr Val
Phe His Arg Ala His 35 40 45Glu Arg Met Leu Phe Gln Thr Ser Tyr Thr
Leu Glu Asn Asn Gly Ser 50 55 60Val Ile Cys Ile Pro Asn Asn Gly Gln
Cys Phe Cys Leu Ala Trp Leu65 70 75 80Lys Ser Asn Gly Thr Asn Ala
Glu Lys Leu Ala Ala Asn Ile Leu Gln 85 90 95Trp Val Thr Phe Ala Leu
Ser Val Ala Cys Leu Gly Trp Tyr Ala Tyr 100 105 110Gln Ala Trp Arg
Ala Thr Cys Gly Trp Glu Glu Val Tyr Val Ala Leu 115 120 125Ile Glu
Met Met Lys Ser Ile Ile Glu Ala Phe His Glu Phe Asp Ser 130 135
140Pro Ala Thr Leu Trp Leu Ser Ser Gly Asn Gly Val Val Trp Met
Arg145 150 155 160Tyr Gly Glu Trp Leu Leu Thr Cys Pro Val Ile Leu
Ile His Leu Ser 165 170 175Asn Leu Thr Gly Leu Lys Asp Asp Tyr Ser
Lys Arg Thr Met Gly Leu 180 185 190Leu Val Ser Asp Val Gly Cys Ile
Val Trp Gly Ala Thr Ser Ala Met 195 200 205Cys Thr Gly Trp Thr Lys
Ile Leu Phe Phe Leu Ile Ser Leu Ser Tyr 210 215 220Gly Met Tyr Thr
Tyr Phe His Ala Ala Lys Val Tyr Ile Glu Ala Phe225 230 235 240His
Thr Val Pro Lys Gly Leu Cys Arg Gln Leu Val Arg Ala Met Ala 245 250
255Trp Leu Phe Phe Val Ser Trp Gly Met Phe Pro Val Leu Phe Leu Leu
260 265 270Gly Pro Glu Gly Phe Gly His Ile Ser Pro Tyr Gly Ser Ala
Ile Gly 275 280 285His Ser Ile Leu Asp Leu Ile Ala Lys Asn Met Trp
Gly Val Leu Gly 290 295 300Asn Tyr Leu Arg Val Lys Ile His Glu His
Ile Leu Leu Tyr Gly Asp305 310 315 320Ile Arg Lys Lys Gln Lys Ile
Thr Ile Ala Gly Gln Glu Met Glu Val 325 330 335Glu Thr Leu Val Ala
Glu Glu Glu Asp Lys Tyr Glu Ser Ser Ala Ala 340 345 350Ala Lys Ser
Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln 355 360 365Ile
Asp Ile Asn Val Phe Cys Tyr Glu Asn Glu Val 370 375
38016316PRTArtificial sequenceSynthetic polypeptide 16Met Gly Gly
Ala Pro Ala Pro Asp Ala His Ser Ala Pro Pro Gly Asn1 5 10 15Asp Ser
Ala Gly Gly Ser Glu Tyr His Ala Pro Ala Gly Tyr Gln Val 20 25 30Asn
Pro Pro Tyr His Pro Val His Gly Tyr Glu Glu Gln Cys Ser Ser 35 40
45Ile Tyr Ile Tyr Tyr Gly Ala Leu Trp Glu Gln Glu Thr Ala Arg Gly
50 55 60Phe Gln Trp Phe Ala Val Phe Leu Ser Ala Leu Phe Leu Ala Phe
Tyr65 70 75 80Gly Trp His Ala Tyr Lys Ala Ser Val Gly Trp Glu Glu
Val Tyr Val 85 90 95Cys Ser Val Glu Leu Ile Lys Val Ile Leu Glu Ile
Tyr Phe Glu Phe 100 105 110Thr Ser Pro Ala Met Leu Phe Leu Tyr Gly
Gly Asn Ile Thr Pro Trp 115 120
125Leu Arg Tyr Ala Glu Trp Leu Leu Thr Cys Pro Val Ile Leu Ile His
130 135 140Leu Ser Asn Ile Thr Gly Leu Ser Glu Glu Tyr Asn Lys Arg
Thr Met145 150 155 160Ala Leu Leu Val Ser Asp Leu Gly Thr Ile Cys
Met Gly Val Thr Ala 165 170 175Ala Leu Ala Thr Gly Trp Val Lys Trp
Leu Phe Tyr Cys Ile Gly Leu 180 185 190Val Tyr Gly Thr Gln Thr Phe
Tyr Asn Ala Gly Ile Ile Tyr Val Glu 195 200 205Ser Tyr Tyr Ile Met
Pro Ala Gly Gly Cys Lys Lys Leu Val Leu Ala 210 215 220Met Thr Ala
Val Tyr Tyr Ser Ser Trp Leu Met Phe Pro Gly Leu Phe225 230 235
240Ile Phe Gly Pro Glu Gly Met His Thr Leu Ser Val Ala Gly Ser Thr
245 250 255Ile Gly His Thr Ile Ala Asp Leu Leu Ser Lys Asn Ile Trp
Gly Leu 260 265 270Leu Gly His Phe Leu Arg Ile Lys Ile His Glu His
Ile Ile Met Tyr 275 280 285Gly Asp Ile Arg Arg Pro Val Ser Ser Gln
Phe Leu Gly Arg Lys Val 290 295 300Asp Val Leu Ala Phe Val Thr Glu
Glu Asp Lys Val305 310 31517346PRTArtificial sequenceSynthetic
polypeptide 17Met Gly Gly Ala Pro Ala Pro Asp Ala His Ser Ala Pro
Pro Gly Asn1 5 10 15Asp Ser Ala Gly Gly Ser Glu Tyr His Ala Pro Ala
Gly Tyr Gln Val 20 25 30Asn Pro Pro Tyr His Pro Val His Gly Tyr Glu
Glu Gln Cys Ser Ser 35 40 45Ile Tyr Ile Tyr Tyr Gly Ala Leu Trp Glu
Gln Glu Thr Ala Arg Gly 50 55 60Phe Gln Trp Phe Ala Val Phe Leu Ser
Ala Leu Phe Leu Ala Phe Tyr65 70 75 80Gly Trp His Ala Tyr Lys Ala
Ser Val Gly Trp Glu Glu Val Tyr Val 85 90 95Cys Ser Val Glu Leu Ile
Lys Val Ile Leu Glu Ile Tyr Phe Glu Phe 100 105 110Thr Ser Pro Ala
Met Leu Phe Leu Tyr Gly Gly Asn Ile Thr Pro Trp 115 120 125Leu Arg
Tyr Ala Glu Trp Leu Leu Thr Cys Pro Val Ile Leu Ile His 130 135
140Leu Ser Asn Ile Thr Gly Leu Ser Glu Glu Tyr Asn Lys Arg Thr
Met145 150 155 160Ala Leu Leu Val Ser Asp Leu Gly Thr Ile Cys Met
Gly Val Thr Ala 165 170 175Ala Leu Ala Thr Gly Trp Val Lys Trp Leu
Phe Tyr Cys Ile Gly Leu 180 185 190Val Tyr Gly Thr Gln Thr Phe Tyr
Asn Ala Gly Ile Ile Tyr Val Glu 195 200 205Ser Tyr Tyr Ile Met Pro
Ala Gly Gly Cys Lys Lys Leu Val Leu Ala 210 215 220Met Thr Ala Val
Tyr Tyr Ser Ser Trp Leu Met Phe Pro Gly Leu Phe225 230 235 240Ile
Phe Gly Pro Glu Gly Met His Thr Leu Ser Val Ala Gly Ser Thr 245 250
255Ile Gly His Thr Ile Ala Asp Leu Leu Ser Lys Asn Ile Trp Gly Leu
260 265 270Leu Gly His Phe Leu Arg Ile Lys Ile His Glu His Ile Ile
Met Tyr 275 280 285Gly Asp Ile Arg Arg Pro Val Ser Ser Gln Phe Leu
Gly Arg Lys Val 290 295 300Asp Val Leu Ala Phe Val Thr Glu Glu Asp
Lys Val Ala Ala Ala Lys305 310 315 320Ser Arg Ile Thr Ser Glu Gly
Glu Tyr Ile Pro Leu Asp Gln Ile Asp 325 330 335Ile Asn Val Phe Cys
Tyr Glu Asn Glu Val 340 34518350PRTArtificial sequenceSynthetic
polypeptide 18Met Ala Glu Leu Ile Ser Ser Ala Thr Arg Ser Leu Phe
Ala Ala Gly1 5 10 15Gly Ile Asn Pro Trp Pro Asn Pro Tyr His His Glu
Asp Met Gly Cys 20 25 30Gly Gly Met Thr Pro Thr Gly Glu Cys Phe Ser
Thr Glu Trp Trp Cys 35 40 45Asp Pro Ser Tyr Gly Leu Ser Asp Ala Gly
Tyr Gly Tyr Cys Phe Val 50 55 60Glu Ala Thr Gly Gly Tyr Leu Val Val
Gly Val Glu Lys Lys Gln Ala65 70 75 80Trp Leu His Ser Arg Gly Thr
Pro Gly Glu Lys Ile Gly Ala Gln Val 85 90 95Cys Gln Trp Ile Ala Phe
Ser Ile Ala Ile Ala Leu Leu Thr Phe Tyr 100 105 110Gly Phe Ser Ala
Trp Lys Ala Thr Cys Gly Trp Glu Glu Val Tyr Val 115 120 125Cys Cys
Val Glu Val Leu Phe Val Thr Leu Glu Ile Phe Lys Glu Phe 130 135
140Ser Ser Pro Ala Thr Val Tyr Leu Ser Thr Gly Asn His Ala Tyr
Cys145 150 155 160Leu Arg Tyr Phe Glu Trp Leu Leu Ser Cys Pro Val
Ile Leu Ile Lys 165 170 175Leu Ser Asn Leu Ser Gly Leu Lys Asn Asp
Tyr Ser Lys Arg Thr Met 180 185 190Gly Leu Ile Val Ser Cys Val Gly
Met Ile Val Phe Gly Met Ala Ala 195 200 205Gly Leu Ala Thr Asp Trp
Leu Lys Trp Leu Leu Tyr Ile Val Ser Cys 210 215 220Ile Tyr Gly Gly
Tyr Met Tyr Phe Gln Ala Ala Lys Cys Tyr Val Glu225 230 235 240Ala
Asn His Ser Val Pro Lys Gly His Cys Arg Met Val Val Lys Leu 245 250
255Met Ala Tyr Ala Tyr Phe Ala Ser Trp Gly Ser Tyr Pro Ile Leu Trp
260 265 270Ala Val Gly Pro Glu Gly Leu Leu Lys Leu Ser Pro Tyr Ala
Asn Ser 275 280 285Ile Gly His Ser Ile Cys Asp Ile Ile Ala Lys Glu
Phe Trp Thr Phe 290 295 300Leu Ala His His Leu Arg Ile Lys Ile His
Glu His Ile Leu Ile His305 310 315 320Gly Asp Ile Arg Lys Thr Thr
Lys Met Glu Ile Gly Gly Glu Glu Val 325 330 335Glu Val Glu Glu Phe
Val Glu Glu Glu Asp Glu Asp Thr Val 340 345 35019380PRTArtificial
sequenceSynthetic polypeptide 19Met Ala Glu Leu Ile Ser Ser Ala Thr
Arg Ser Leu Phe Ala Ala Gly1 5 10 15Gly Ile Asn Pro Trp Pro Asn Pro
Tyr His His Glu Asp Met Gly Cys 20 25 30Gly Gly Met Thr Pro Thr Gly
Glu Cys Phe Ser Thr Glu Trp Trp Cys 35 40 45Asp Pro Ser Tyr Gly Leu
Ser Asp Ala Gly Tyr Gly Tyr Cys Phe Val 50 55 60Glu Ala Thr Gly Gly
Tyr Leu Val Val Gly Val Glu Lys Lys Gln Ala65 70 75 80Trp Leu His
Ser Arg Gly Thr Pro Gly Glu Lys Ile Gly Ala Gln Val 85 90 95Cys Gln
Trp Ile Ala Phe Ser Ile Ala Ile Ala Leu Leu Thr Phe Tyr 100 105
110Gly Phe Ser Ala Trp Lys Ala Thr Cys Gly Trp Glu Glu Val Tyr Val
115 120 125Cys Cys Val Glu Val Leu Phe Val Thr Leu Glu Ile Phe Lys
Glu Phe 130 135 140Ser Ser Pro Ala Thr Val Tyr Leu Ser Thr Gly Asn
His Ala Tyr Cys145 150 155 160Leu Arg Tyr Phe Glu Trp Leu Leu Ser
Cys Pro Val Ile Leu Ile Lys 165 170 175Leu Ser Asn Leu Ser Gly Leu
Lys Asn Asp Tyr Ser Lys Arg Thr Met 180 185 190Gly Leu Ile Val Ser
Cys Val Gly Met Ile Val Phe Gly Met Ala Ala 195 200 205Gly Leu Ala
Thr Asp Trp Leu Lys Trp Leu Leu Tyr Ile Val Ser Cys 210 215 220Ile
Tyr Gly Gly Tyr Met Tyr Phe Gln Ala Ala Lys Cys Tyr Val Glu225 230
235 240Ala Asn His Ser Val Pro Lys Gly His Cys Arg Met Val Val Lys
Leu 245 250 255Met Ala Tyr Ala Tyr Phe Ala Ser Trp Gly Ser Tyr Pro
Ile Leu Trp 260 265 270Ala Val Gly Pro Glu Gly Leu Leu Lys Leu Ser
Pro Tyr Ala Asn Ser 275 280 285Ile Gly His Ser Ile Cys Asp Ile Ile
Ala Lys Glu Phe Trp Thr Phe 290 295 300Leu Ala His His Leu Arg Ile
Lys Ile His Glu His Ile Leu Ile His305 310 315 320Gly Asp Ile Arg
Lys Thr Thr Lys Met Glu Ile Gly Gly Glu Glu Val 325 330 335Glu Val
Glu Glu Phe Val Glu Glu Glu Asp Glu Asp Thr Val Ala Ala 340 345
350Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln
355 360 365Ile Asp Ile Asn Val Phe Cys Tyr Glu Asn Glu Val 370 375
38020345PRTArtificial sequenceSynthetic polypeptide 20Met Ser Arg
Leu Val Ala Ala Ser Trp Leu Leu Ala Leu Leu Leu Cys1 5 10 15Gly Ile
Thr Ser Thr Thr Thr Ala Ser Ser Ala Pro Ala Ala Ser Ser 20 25 30Thr
Asp Gly Thr Ala Ala Ala Ala Val Ser His Tyr Ala Met Asn Gly 35 40
45Phe Asp Glu Leu Ala Lys Gly Ala Val Val Pro Glu Asp His Phe Val
50 55 60Cys Gly Pro Ala Asp Lys Cys Tyr Cys Ser Ala Trp Leu His Ser
Arg65 70 75 80Gly Thr Pro Gly Glu Lys Ile Gly Ala Gln Val Cys Gln
Trp Ile Ala 85 90 95Phe Ser Ile Ala Ile Ala Leu Leu Thr Phe Tyr Gly
Phe Ser Ala Trp 100 105 110Lys Ala Thr Cys Gly Trp Glu Glu Val Tyr
Val Cys Cys Val Glu Val 115 120 125Leu Phe Val Thr Leu Glu Ile Phe
Lys Glu Phe Ser Ser Pro Ala Thr 130 135 140Val Tyr Leu Ser Thr Gly
Asn His Ala Tyr Cys Leu Arg Tyr Phe Glu145 150 155 160Trp Leu Leu
Ser Cys Pro Val Ile Leu Ile Lys Leu Ser Asn Leu Ser 165 170 175Gly
Leu Lys Asn Asp Tyr Ser Lys Arg Thr Met Gly Leu Ile Val Ser 180 185
190Cys Val Gly Met Ile Val Phe Gly Met Ala Ala Gly Leu Ala Thr Asp
195 200 205Trp Leu Lys Trp Leu Leu Tyr Ile Val Ser Cys Ile Tyr Gly
Gly Tyr 210 215 220Met Tyr Phe Gln Ala Ala Lys Cys Tyr Val Glu Ala
Asn His Ser Val225 230 235 240Pro Lys Gly His Cys Arg Met Val Val
Lys Leu Met Ala Tyr Ala Tyr 245 250 255Phe Ala Ser Trp Gly Ser Tyr
Pro Ile Leu Trp Ala Val Gly Pro Glu 260 265 270Gly Leu Leu Lys Leu
Ser Pro Tyr Ala Asn Ser Ile Gly His Ser Ile 275 280 285Cys Asp Ile
Ile Ala Lys Glu Phe Trp Thr Phe Leu Ala His His Leu 290 295 300Arg
Ile Lys Ile His Glu His Ile Leu Ile His Gly Asp Ile Arg Lys305 310
315 320Thr Thr Lys Met Glu Ile Gly Gly Glu Glu Val Glu Val Glu Glu
Phe 325 330 335Val Glu Glu Glu Asp Glu Asp Thr Val 340
34521375PRTArtificial sequenceSynthetic polypeptide 21Met Ser Arg
Leu Val Ala Ala Ser Trp Leu Leu Ala Leu Leu Leu Cys1 5 10 15Gly Ile
Thr Ser Thr Thr Thr Ala Ser Ser Ala Pro Ala Ala Ser Ser 20 25 30Thr
Asp Gly Thr Ala Ala Ala Ala Val Ser His Tyr Ala Met Asn Gly 35 40
45Phe Asp Glu Leu Ala Lys Gly Ala Val Val Pro Glu Asp His Phe Val
50 55 60Cys Gly Pro Ala Asp Lys Cys Tyr Cys Ser Ala Trp Leu His Ser
Arg65 70 75 80Gly Thr Pro Gly Glu Lys Ile Gly Ala Gln Val Cys Gln
Trp Ile Ala 85 90 95Phe Ser Ile Ala Ile Ala Leu Leu Thr Phe Tyr Gly
Phe Ser Ala Trp 100 105 110Lys Ala Thr Cys Gly Trp Glu Glu Val Tyr
Val Cys Cys Val Glu Val 115 120 125Leu Phe Val Thr Leu Glu Ile Phe
Lys Glu Phe Ser Ser Pro Ala Thr 130 135 140Val Tyr Leu Ser Thr Gly
Asn His Ala Tyr Cys Leu Arg Tyr Phe Glu145 150 155 160Trp Leu Leu
Ser Cys Pro Val Ile Leu Ile Lys Leu Ser Asn Leu Ser 165 170 175Gly
Leu Lys Asn Asp Tyr Ser Lys Arg Thr Met Gly Leu Ile Val Ser 180 185
190Cys Val Gly Met Ile Val Phe Gly Met Ala Ala Gly Leu Ala Thr Asp
195 200 205Trp Leu Lys Trp Leu Leu Tyr Ile Val Ser Cys Ile Tyr Gly
Gly Tyr 210 215 220Met Tyr Phe Gln Ala Ala Lys Cys Tyr Val Glu Ala
Asn His Ser Val225 230 235 240Pro Lys Gly His Cys Arg Met Val Val
Lys Leu Met Ala Tyr Ala Tyr 245 250 255Phe Ala Ser Trp Gly Ser Tyr
Pro Ile Leu Trp Ala Val Gly Pro Glu 260 265 270Gly Leu Leu Lys Leu
Ser Pro Tyr Ala Asn Ser Ile Gly His Ser Ile 275 280 285Cys Asp Ile
Ile Ala Lys Glu Phe Trp Thr Phe Leu Ala His His Leu 290 295 300Arg
Ile Lys Ile His Glu His Ile Leu Ile His Gly Asp Ile Arg Lys305 310
315 320Thr Thr Lys Met Glu Ile Gly Gly Glu Glu Val Glu Val Glu Glu
Phe 325 330 335Val Glu Glu Glu Asp Glu Asp Thr Val Ala Ala Ala Lys
Ser Arg Ile 340 345 350Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln
Ile Asp Ile Asn Val 355 360 365Phe Cys Tyr Glu Asn Glu Val 370
37522325PRTArtificial sequenceSynthetic polypeptide 22Met Glu Thr
Ala Ala Thr Met Thr His Ala Phe Ile Ser Ala Val Pro1 5 10 15Ser Ala
Glu Ala Thr Ile Arg Gly Leu Leu Ser Ala Ala Ala Val Val 20 25 30Thr
Pro Ala Ala Asp Ala His Gly Glu Thr Ser Asn Ala Thr Thr Ala 35 40
45Gly Ala Asp His Gly Cys Phe Pro His Ile Asn His Gly Thr Glu Leu
50 55 60Gln His Lys Ile Ala Val Gly Leu Gln Trp Phe Thr Val Ile Val
Ala65 70 75 80Ile Val Gln Leu Ile Phe Tyr Gly Trp His Ser Phe Lys
Ala Thr Thr 85 90 95Gly Trp Glu Glu Val Tyr Val Cys Val Ile Glu Leu
Val Lys Cys Phe 100 105 110Ile Glu Leu Phe His Glu Val Asp Ser Pro
Ala Thr Val Tyr Gln Thr 115 120 125Asn Gly Gly Ala Val Ile Trp Leu
Arg Tyr Ser Met Trp Leu Leu Thr 130 135 140Cys Pro Val Ile Leu Ile
His Leu Ser Asn Leu Thr Gly Leu His Glu145 150 155 160Glu Tyr Ser
Lys Arg Thr Met Thr Ile Leu Val Thr Asp Ile Gly Asn 165 170 175Ile
Val Trp Gly Ile Thr Ala Ala Phe Thr Lys Gly Pro Leu Lys Ile 180 185
190Leu Phe Phe Met Ile Gly Leu Phe Tyr Gly Val Thr Cys Phe Phe Gln
195 200 205Ile Ala Lys Val Tyr Ile Glu Ser Tyr His Thr Leu Pro Lys
Gly Val 210 215 220Cys Arg Lys Ile Cys Lys Ile Met Ala Tyr Val Phe
Phe Cys Ser Trp225 230 235 240Leu Met Phe Pro Val Met Phe Ile Ala
Gly His Glu Gly Leu Gly Leu 245 250 255Ile Thr Pro Tyr Thr Ser Gly
Ile Gly His Leu Ile Leu Asp Leu Ile 260 265 270Ser Lys Asn Thr Trp
Gly Phe Leu Gly His His Leu Arg Val Lys Ile 275 280 285His Glu His
Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr Thr Ile 290 295 300Asn
Val Ala Gly Glu Asn Met Glu Ile Glu Thr Phe Val Asp Glu Glu305 310
315 320Glu Glu Gly Gly Val 32523355PRTArtificial sequenceSynthetic
polypeptide 23Met Glu Thr Ala Ala Thr Met Thr His Ala Phe Ile Ser
Ala Val Pro1 5 10 15Ser Ala Glu Ala Thr Ile Arg Gly Leu Leu Ser Ala
Ala Ala Val Val 20 25 30Thr Pro Ala Ala Asp Ala His Gly Glu Thr Ser
Asn Ala Thr Thr Ala 35 40 45Gly Ala Asp His Gly Cys Phe Pro His Ile
Asn His Gly Thr Glu Leu 50 55 60Gln His Lys Ile Ala Val Gly Leu Gln
Trp Phe Thr Val Ile Val Ala65 70 75 80Ile Val Gln Leu Ile Phe Tyr
Gly Trp His Ser Phe Lys Ala Thr Thr 85 90 95Gly Trp Glu Glu Val Tyr
Val Cys Val Ile Glu Leu Val Lys Cys Phe 100 105 110Ile Glu Leu Phe
His
Glu Val Asp Ser Pro Ala Thr Val Tyr Gln Thr 115 120 125Asn Gly Gly
Ala Val Ile Trp Leu Arg Tyr Ser Met Trp Leu Leu Thr 130 135 140Cys
Pro Val Ile Leu Ile His Leu Ser Asn Leu Thr Gly Leu His Glu145 150
155 160Glu Tyr Ser Lys Arg Thr Met Thr Ile Leu Val Thr Asp Ile Gly
Asn 165 170 175Ile Val Trp Gly Ile Thr Ala Ala Phe Thr Lys Gly Pro
Leu Lys Ile 180 185 190Leu Phe Phe Met Ile Gly Leu Phe Tyr Gly Val
Thr Cys Phe Phe Gln 195 200 205Ile Ala Lys Val Tyr Ile Glu Ser Tyr
His Thr Leu Pro Lys Gly Val 210 215 220Cys Arg Lys Ile Cys Lys Ile
Met Ala Tyr Val Phe Phe Cys Ser Trp225 230 235 240Leu Met Phe Pro
Val Met Phe Ile Ala Gly His Glu Gly Leu Gly Leu 245 250 255Ile Thr
Pro Tyr Thr Ser Gly Ile Gly His Leu Ile Leu Asp Leu Ile 260 265
270Ser Lys Asn Thr Trp Gly Phe Leu Gly His His Leu Arg Val Lys Ile
275 280 285His Glu His Ile Leu Ile His Gly Asp Ile Arg Lys Thr Thr
Thr Ile 290 295 300Asn Val Ala Gly Glu Asn Met Glu Ile Glu Thr Phe
Val Asp Glu Glu305 310 315 320Glu Glu Gly Gly Val Ala Ala Ala Lys
Ser Arg Ile Thr Ser Glu Gly 325 330 335Glu Tyr Ile Pro Leu Asp Gln
Ile Asp Ile Asn Val Phe Cys Tyr Glu 340 345 350Asn Glu Val
35524258PRTArtificial sequenceSynthetic polypeptide 24Met Asp Pro
Ile Ala Leu Gln Ala Gly Tyr Asp Leu Leu Gly Asp Gly1 5 10 15Arg Pro
Glu Thr Leu Trp Leu Gly Ile Gly Thr Leu Leu Met Leu Ile 20 25 30Gly
Thr Phe Tyr Phe Leu Val Arg Gly Trp Gly Val Thr Asp Lys Asp 35 40
45Ala Arg Glu Tyr Tyr Ala Val Thr Ile Leu Val Pro Gly Ile Ala Ser
50 55 60Ala Ala Tyr Leu Ser Met Phe Phe Gly Ile Gly Leu Thr Glu Val
Thr65 70 75 80Val Gly Gly Glu Met Leu Asp Ile Tyr Tyr Ala Arg Tyr
Ala Asp Trp 85 90 95Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala
Leu Leu Ala Lys 100 105 110Val Asp Arg Val Thr Ile Gly Thr Leu Val
Gly Val Asp Ala Leu Met 115 120 125Ile Val Thr Gly Leu Ile Gly Ala
Leu Ser His Thr Ala Ile Ala Arg 130 135 140Tyr Ser Trp Trp Leu Phe
Ser Thr Ile Cys Met Ile Val Val Leu Tyr145 150 155 160Phe Leu Ala
Thr Ser Leu Arg Ser Ala Ala Lys Glu Arg Gly Pro Glu 165 170 175Val
Ala Ser Thr Phe Asn Thr Leu Thr Ala Leu Val Leu Val Leu Trp 180 185
190Thr Ala Tyr Pro Ile Leu Trp Ile Ile Gly Thr Glu Gly Ala Gly Val
195 200 205Val Gly Leu Gly Ile Glu Thr Leu Leu Phe Met Val Leu Asp
Val Thr 210 215 220Ala Lys Val Gly Phe Gly Phe Ile Leu Leu Arg Ser
Arg Ala Ile Leu225 230 235 240Gly Asp Thr Glu Ala Pro Glu Pro Ser
Ala Gly Ala Asp Val Ser Ala 245 250 255Ala Asp25293PRTArtificial
sequenceSynthetic polypeptide 25Met Asp Pro Ile Ala Leu Gln Ala Gly
Tyr Asp Leu Leu Gly Asp Gly1 5 10 15Arg Pro Glu Thr Leu Trp Leu Gly
Ile Gly Thr Leu Leu Met Leu Ile 20 25 30Gly Thr Phe Tyr Phe Leu Val
Arg Gly Trp Gly Val Thr Asp Lys Asp 35 40 45Ala Arg Glu Tyr Tyr Ala
Val Thr Ile Leu Val Pro Gly Ile Ala Ser 50 55 60Ala Ala Tyr Leu Ser
Met Phe Phe Gly Ile Gly Leu Thr Glu Val Thr65 70 75 80Val Gly Gly
Glu Met Leu Asp Ile Tyr Tyr Ala Arg Tyr Ala Asp Trp 85 90 95Leu Phe
Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala Leu Leu Ala Lys 100 105
110Val Asp Arg Val Thr Ile Gly Thr Leu Val Gly Val Asp Ala Leu Met
115 120 125Ile Val Thr Gly Leu Ile Gly Ala Leu Ser His Thr Ala Ile
Ala Arg 130 135 140Tyr Ser Trp Trp Leu Phe Ser Thr Ile Cys Met Ile
Val Val Leu Tyr145 150 155 160Phe Leu Ala Thr Ser Leu Arg Ser Ala
Ala Lys Glu Arg Gly Pro Glu 165 170 175Val Ala Ser Thr Phe Asn Thr
Leu Thr Ala Leu Val Leu Val Leu Trp 180 185 190Thr Ala Tyr Pro Ile
Leu Trp Ile Ile Gly Thr Glu Gly Ala Gly Val 195 200 205Val Gly Leu
Gly Ile Glu Thr Leu Leu Phe Met Val Leu Asp Val Thr 210 215 220Ala
Lys Val Gly Phe Gly Phe Ile Leu Leu Arg Ser Arg Ala Ile Leu225 230
235 240Gly Asp Thr Glu Ala Pro Glu Pro Ser Ala Gly Ala Asp Val Ser
Ala 245 250 255Ala Asp Arg Pro Val Val Ala Ala Ala Ala Lys Ser Arg
Ile Thr Ser 260 265 270Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile Asp
Ile Asn Val Phe Cys 275 280 285Tyr Glu Asn Glu Val
29026248PRTArtificial sequenceSynthetic polypeptide 26Met Asp Pro
Ile Ala Leu Gln Ala Gly Tyr Asp Leu Leu Gly Asp Gly1 5 10 15Arg Pro
Glu Thr Leu Trp Leu Gly Ile Gly Thr Leu Leu Met Leu Ile 20 25 30Gly
Thr Phe Tyr Phe Ile Val Lys Gly Trp Gly Val Thr Asp Lys Glu 35 40
45Ala Arg Glu Tyr Tyr Ser Ile Thr Ile Leu Val Pro Gly Ile Ala Ser
50 55 60Ala Ala Tyr Leu Ser Met Phe Phe Gly Ile Gly Leu Thr Glu Val
Thr65 70 75 80Val Ala Gly Glu Val Leu Asp Ile Tyr Tyr Ala Arg Tyr
Ala Asp Trp 85 90 95Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala
Leu Leu Ala Lys 100 105 110Val Asp Arg Val Ser Ile Gly Thr Leu Val
Gly Val Asp Ala Leu Met 115 120 125Ile Val Thr Gly Leu Ile Gly Ala
Leu Ser His Thr Pro Leu Ala Arg 130 135 140Tyr Ser Trp Trp Leu Phe
Ser Thr Ile Cys Met Ile Val Val Leu Tyr145 150 155 160Phe Leu Ala
Thr Ser Leu Arg Ala Ala Ala Lys Glu Arg Gly Pro Glu 165 170 175Val
Ala Ser Thr Phe Asn Thr Leu Thr Ala Leu Val Leu Val Leu Trp 180 185
190Thr Ala Tyr Pro Ile Leu Trp Ile Ile Gly Thr Glu Gly Ala Gly Val
195 200 205Val Gly Leu Gly Ile Glu Thr Leu Leu Phe Met Val Leu Asp
Val Thr 210 215 220Ala Lys Val Gly Phe Gly Phe Ile Leu Leu Arg Ser
Arg Ala Ile Leu225 230 235 240Gly Asp Thr Glu Ala Pro Glu Pro
24527278PRTArtificial sequenceSynthetic polypeptide 27Met Asp Pro
Ile Ala Leu Gln Ala Gly Tyr Asp Leu Leu Gly Asp Gly1 5 10 15Arg Pro
Glu Thr Leu Trp Leu Gly Ile Gly Thr Leu Leu Met Leu Ile 20 25 30Gly
Thr Phe Tyr Phe Ile Val Lys Gly Trp Gly Val Thr Asp Lys Glu 35 40
45Ala Arg Glu Tyr Tyr Ser Ile Thr Ile Leu Val Pro Gly Ile Ala Ser
50 55 60Ala Ala Tyr Leu Ser Met Phe Phe Gly Ile Gly Leu Thr Glu Val
Thr65 70 75 80Val Ala Gly Glu Val Leu Asp Ile Tyr Tyr Ala Arg Tyr
Ala Asp Trp 85 90 95Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu Ala
Leu Leu Ala Lys 100 105 110Val Asp Arg Val Ser Ile Gly Thr Leu Val
Gly Val Asp Ala Leu Met 115 120 125Ile Val Thr Gly Leu Ile Gly Ala
Leu Ser His Thr Pro Leu Ala Arg 130 135 140Tyr Ser Trp Trp Leu Phe
Ser Thr Ile Cys Met Ile Val Val Leu Tyr145 150 155 160Phe Leu Ala
Thr Ser Leu Arg Ala Ala Ala Lys Glu Arg Gly Pro Glu 165 170 175Val
Ala Ser Thr Phe Asn Thr Leu Thr Ala Leu Val Leu Val Leu Trp 180 185
190Thr Ala Tyr Pro Ile Leu Trp Ile Ile Gly Thr Glu Gly Ala Gly Val
195 200 205Val Gly Leu Gly Ile Glu Thr Leu Leu Phe Met Val Leu Asp
Val Thr 210 215 220Ala Lys Val Gly Phe Gly Phe Ile Leu Leu Arg Ser
Arg Ala Ile Leu225 230 235 240Gly Asp Thr Glu Ala Pro Glu Pro Ala
Ala Ala Lys Ser Arg Ile Thr 245 250 255Ser Glu Gly Glu Tyr Ile Pro
Leu Asp Gln Ile Asp Ile Asn Val Phe 260 265 270Cys Tyr Glu Asn Glu
Val 27528242PRTArtificial sequenceSynthetic polypeptide 28Met Leu
Val Gly Glu Gly Ala Lys Leu Asp Val His Gly Cys Lys Thr1 5 10 15Val
Asp Met Ala Ser Ser Phe Gly Lys Ala Leu Leu Glu Phe Val Phe 20 25
30Ile Val Phe Ala Cys Ile Thr Leu Leu Leu Gly Ile Asn Ala Ala Lys
35 40 45Ser Lys Ala Ala Ser Arg Val Leu Phe Pro Ala Thr Phe Val Thr
Gly 50 55 60Ile Ala Ser Ile Ala Tyr Phe Ser Met Ala Ser Gly Gly Gly
Trp Val65 70 75 80Ile Ala Pro Asp Cys Arg Gln Leu Phe Val Ala Arg
Tyr Leu Asp Trp 85 90 95Leu Ile Thr Thr Pro Leu Leu Leu Ile Asp Leu
Gly Leu Val Ala Gly 100 105 110Val Ser Arg Trp Asp Ile Met Ala Leu
Cys Leu Ser Asp Val Leu Met 115 120 125Ile Ala Thr Gly Ala Phe Gly
Ser Leu Thr Val Gly Asn Val Lys Trp 130 135 140Val Trp Trp Phe Phe
Gly Met Cys Trp Phe Leu His Ile Ile Phe Ala145 150 155 160Leu Gly
Lys Ser Trp Ala Glu Ala Ala Lys Ala Lys Gly Gly Asp Ser 165 170
175Ala Ser Val Tyr Ser Lys Ile Ala Gly Ile Thr Val Ile Thr Trp Phe
180 185 190Cys Tyr Pro Val Val Trp Val Phe Ala Glu Gly Phe Gly Asn
Phe Ser 195 200 205Val Thr Phe Glu Val Leu Ile Tyr Gly Val Leu Asp
Val Ile Ser Lys 210 215 220Ala Val Phe Gly Leu Ile Leu Met Ser Gly
Ala Ala Thr Gly Tyr Glu225 230 235 240Ser Ile29272PRTArtificial
sequenceSynthetic polypeptide 29Met Leu Val Gly Glu Gly Ala Lys Leu
Asp Val His Gly Cys Lys Thr1 5 10 15Val Asp Met Ala Ser Ser Phe Gly
Lys Ala Leu Leu Glu Phe Val Phe 20 25 30Ile Val Phe Ala Cys Ile Thr
Leu Leu Leu Gly Ile Asn Ala Ala Lys 35 40 45Ser Lys Ala Ala Ser Arg
Val Leu Phe Pro Ala Thr Phe Val Thr Gly 50 55 60Ile Ala Ser Ile Ala
Tyr Phe Ser Met Ala Ser Gly Gly Gly Trp Val65 70 75 80Ile Ala Pro
Asp Cys Arg Gln Leu Phe Val Ala Arg Tyr Leu Asp Trp 85 90 95Leu Ile
Thr Thr Pro Leu Leu Leu Ile Asp Leu Gly Leu Val Ala Gly 100 105
110Val Ser Arg Trp Asp Ile Met Ala Leu Cys Leu Ser Asp Val Leu Met
115 120 125Ile Ala Thr Gly Ala Phe Gly Ser Leu Thr Val Gly Asn Val
Lys Trp 130 135 140Val Trp Trp Phe Phe Gly Met Cys Trp Phe Leu His
Ile Ile Phe Ala145 150 155 160Leu Gly Lys Ser Trp Ala Glu Ala Ala
Lys Ala Lys Gly Gly Asp Ser 165 170 175Ala Ser Val Tyr Ser Lys Ile
Ala Gly Ile Thr Val Ile Thr Trp Phe 180 185 190Cys Tyr Pro Val Val
Trp Val Phe Ala Glu Gly Phe Gly Asn Phe Ser 195 200 205Val Thr Phe
Glu Val Leu Ile Tyr Gly Val Leu Asp Val Ile Ser Lys 210 215 220Ala
Val Phe Gly Leu Ile Leu Met Ser Gly Ala Ala Thr Gly Tyr Glu225 230
235 240Ser Ile Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr
Ile 245 250 255Pro Leu Asp Gln Ile Asp Ile Asn Val Phe Cys Tyr Glu
Asn Glu Val 260 265 27030262PRTArtificial sequenceSynthetic
polypeptide 30Met Ala Pro Leu Ala Gln Asp Trp Thr Tyr Ala Glu Trp
Ser Ala Val1 5 10 15Tyr Asn Ala Leu Ser Phe Gly Ile Ala Gly Met Gly
Ser Ala Thr Ile 20 25 30Phe Phe Trp Leu Gln Leu Pro Asn Val Thr Lys
Asn Tyr Arg Thr Ala 35 40 45Leu Thr Ile Thr Gly Ile Val Thr Leu Ile
Ala Thr Tyr His Tyr Phe 50 55 60Arg Ile Phe Asn Ser Trp Val Ala Ala
Phe Asn Val Gly Leu Gly Val65 70 75 80Asn Gly Ala Tyr Glu Val Thr
Val Ser Gly Thr Pro Phe Asn Asp Ala 85 90 95Tyr Arg Tyr Val Asp Trp
Leu Leu Thr Val Pro Leu Leu Leu Val Glu 100 105 110Leu Ile Leu Val
Met Lys Leu Pro Ala Lys Glu Thr Val Cys Leu Ala 115 120 125Trp Thr
Leu Gly Ile Ala Ser Ala Val Met Val Ala Leu Gly Tyr Pro 130 135
140Gly Glu Ile Gln Asp Asp Leu Ser Val Arg Trp Phe Trp Trp Ala
Cys145 150 155 160Ala Met Val Pro Phe Val Tyr Val Val Gly Thr Leu
Val Val Gly Leu 165 170 175Gly Ala Ala Thr Ala Lys Gln Pro Glu Gly
Val Val Asp Leu Val Ser 180 185 190Ala Ala Arg Tyr Leu Thr Val Val
Ser Trp Leu Thr Tyr Pro Phe Val 195 200 205Tyr Ile Val Lys Asn Ile
Gly Leu Ala Gly Ser Thr Ala Thr Met Tyr 210 215 220Glu Gln Ile Gly
Tyr Ser Ala Ala Asp Val Thr Ala Lys Ala Val Phe225 230 235 240Gly
Val Leu Ile Trp Ala Ile Ala Asn Ala Lys Ser Arg Leu Glu Glu 245 250
255Glu Gly Lys Leu Arg Ala 26031292PRTArtificial sequenceSynthetic
polypeptide 31Met Ala Pro Leu Ala Gln Asp Trp Thr Tyr Ala Glu Trp
Ser Ala Val1 5 10 15Tyr Asn Ala Leu Ser Phe Gly Ile Ala Gly Met Gly
Ser Ala Thr Ile 20 25 30Phe Phe Trp Leu Gln Leu Pro Asn Val Thr Lys
Asn Tyr Arg Thr Ala 35 40 45Leu Thr Ile Thr Gly Ile Val Thr Leu Ile
Ala Thr Tyr His Tyr Phe 50 55 60Arg Ile Phe Asn Ser Trp Val Ala Ala
Phe Asn Val Gly Leu Gly Val65 70 75 80Asn Gly Ala Tyr Glu Val Thr
Val Ser Gly Thr Pro Phe Asn Asp Ala 85 90 95Tyr Arg Tyr Val Asp Trp
Leu Leu Thr Val Pro Leu Leu Leu Val Glu 100 105 110Leu Ile Leu Val
Met Lys Leu Pro Ala Lys Glu Thr Val Cys Leu Ala 115 120 125Trp Thr
Leu Gly Ile Ala Ser Ala Val Met Val Ala Leu Gly Tyr Pro 130 135
140Gly Glu Ile Gln Asp Asp Leu Ser Val Arg Trp Phe Trp Trp Ala
Cys145 150 155 160Ala Met Val Pro Phe Val Tyr Val Val Gly Thr Leu
Val Val Gly Leu 165 170 175Gly Ala Ala Thr Ala Lys Gln Pro Glu Gly
Val Val Asp Leu Val Ser 180 185 190Ala Ala Arg Tyr Leu Thr Val Val
Ser Trp Leu Thr Tyr Pro Phe Val 195 200 205Tyr Ile Val Lys Asn Ile
Gly Leu Ala Gly Ser Thr Ala Thr Met Tyr 210 215 220Glu Gln Ile Gly
Tyr Ser Ala Ala Asp Val Thr Ala Lys Ala Val Phe225 230 235 240Gly
Val Leu Ile Trp Ala Ile Ala Asn Ala Lys Ser Arg Leu Glu Glu 245 250
255Glu Gly Lys Leu Arg Ala Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu
260 265 270Gly Glu Tyr Ile Pro Leu Asp Gln Ile Asp Ile Asn Val Phe
Cys Tyr 275 280 285Glu Asn Glu Val 29032313PRTArtificial
sequenceSynthetic polypeptide 32Met Ile Val Asp Gln Phe Glu Glu Val
Leu Met Lys Thr Ser Gln Leu1 5 10 15Phe Pro Leu Pro Thr Ala Thr Gln
Ser Ala Gln Pro Thr His Val Ala
20 25 30Pro Val Pro Thr Val Leu Pro Asp Thr Pro Ile Tyr Glu Thr Val
Gly 35 40 45Asp Ser Gly Ser Lys Thr Leu Trp Val Val Phe Val Leu Met
Leu Ile 50 55 60Ala Ser Ala Ala Phe Thr Ala Leu Ser Trp Lys Ile Pro
Val Asn Arg65 70 75 80Arg Leu Tyr His Val Ile Thr Thr Ile Ile Thr
Leu Thr Ala Ala Leu 85 90 95Ser Tyr Phe Ala Met Ala Thr Gly His Gly
Val Ala Leu Asn Lys Ile 100 105 110Val Ile Arg Thr Gln His Asp His
Val Pro Asp Thr Tyr Glu Thr Val 115 120 125Tyr Arg Gln Val Tyr Tyr
Ala Arg Tyr Ile Asp Trp Ala Ile Thr Thr 130 135 140Pro Leu Leu Leu
Leu Asp Leu Gly Leu Leu Ala Gly Met Ser Gly Ala145 150 155 160His
Ile Phe Met Ala Ile Val Ala Asp Leu Ile Met Val Leu Thr Gly 165 170
175Leu Phe Ala Ala Phe Gly Ser Glu Gly Thr Pro Gln Lys Trp Gly Trp
180 185 190Tyr Thr Ile Ala Cys Ile Ala Tyr Ile Phe Val Val Trp His
Leu Val 195 200 205Leu Asn Gly Gly Ala Asn Ala Arg Val Lys Gly Glu
Lys Leu Arg Ser 210 215 220Phe Phe Val Ala Ile Gly Ala Tyr Thr Leu
Ile Leu Trp Thr Ala Tyr225 230 235 240Pro Ile Val Trp Gly Leu Ala
Asp Gly Ala Arg Lys Ile Gly Val Asp 245 250 255Gly Glu Ile Ile Ala
Tyr Ala Val Leu Asp Val Leu Ala Lys Gly Val 260 265 270Phe Gly Ala
Trp Leu Leu Val Thr His Ala Asn Leu Arg Glu Ser Asp 275 280 285Val
Glu Leu Asn Gly Phe Trp Ala Asn Gly Leu Asn Arg Glu Gly Ala 290 295
300Ile Arg Ile Gly Glu Asp Asp Gly Ala305 31033351PRTArtificial
sequenceSynthetic polypeptide 33Met Ile Val Asp Gln Phe Glu Glu Val
Leu Met Lys Thr Ser Gln Leu1 5 10 15Phe Pro Leu Pro Thr Ala Thr Gln
Ser Ala Gln Pro Thr His Val Ala 20 25 30Pro Val Pro Thr Val Leu Pro
Asp Thr Pro Ile Tyr Glu Thr Val Gly 35 40 45Asp Ser Gly Ser Lys Thr
Leu Trp Val Val Phe Val Leu Met Leu Ile 50 55 60Ala Ser Ala Ala Phe
Thr Ala Leu Ser Trp Lys Ile Pro Val Asn Arg65 70 75 80Arg Leu Tyr
His Val Ile Thr Thr Ile Ile Thr Leu Thr Ala Ala Leu 85 90 95Ser Tyr
Phe Ala Met Ala Thr Gly His Gly Val Ala Leu Asn Lys Ile 100 105
110Val Ile Arg Thr Gln His Asp His Val Pro Asp Thr Tyr Glu Thr Val
115 120 125Tyr Arg Gln Val Tyr Tyr Ala Arg Tyr Ile Asp Trp Ala Ile
Thr Thr 130 135 140Pro Leu Leu Leu Leu Asp Leu Gly Leu Leu Ala Gly
Met Ser Gly Ala145 150 155 160His Ile Phe Met Ala Ile Val Ala Asp
Leu Ile Met Val Leu Thr Gly 165 170 175Leu Phe Ala Ala Phe Gly Ser
Glu Gly Thr Pro Gln Lys Trp Gly Trp 180 185 190Tyr Thr Ile Ala Cys
Ile Ala Tyr Ile Phe Val Val Trp His Leu Val 195 200 205Leu Asn Gly
Gly Ala Asn Ala Arg Val Lys Gly Glu Lys Leu Arg Ser 210 215 220Phe
Phe Val Ala Ile Gly Ala Tyr Thr Leu Ile Leu Trp Thr Ala Tyr225 230
235 240Pro Ile Val Trp Gly Leu Ala Asp Gly Ala Arg Lys Ile Gly Val
Asp 245 250 255Gly Glu Ile Ile Ala Tyr Ala Val Leu Asp Val Leu Ala
Lys Gly Val 260 265 270Phe Gly Ala Trp Leu Leu Val Thr His Ala Asn
Leu Arg Glu Ser Asp 275 280 285Val Glu Leu Asn Gly Phe Trp Ala Asn
Gly Leu Asn Arg Glu Gly Ala 290 295 300Ile Arg Ile Gly Glu Asp Asp
Gly Ala Arg Pro Val Val Ala Val Ser305 310 315 320Lys Ala Ala Ala
Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro 325 330 335Leu Asp
Gln Ile Asp Ile Asn Val Phe Cys Tyr Glu Asn Glu Val 340 345
35034291PRTArtificial sequenceSynthetic polypeptide 34Met Thr Glu
Thr Leu Pro Pro Val Thr Glu Ser Ala Val Ala Leu Gln1 5 10 15Ala Glu
Val Thr Gln Arg Glu Leu Phe Glu Phe Val Leu Asn Asp Pro 20 25 30Leu
Leu Ala Ser Ser Leu Tyr Ile Asn Ile Ala Leu Ala Gly Leu Ser 35 40
45Ile Leu Leu Phe Val Phe Met Thr Arg Gly Leu Asp Asp Pro Arg Ala
50 55 60Lys Leu Ile Ala Val Ser Thr Ile Leu Val Pro Val Val Ser Ile
Ala65 70 75 80Ser Tyr Thr Gly Leu Ala Ser Gly Leu Thr Ile Ser Val
Leu Glu Met 85 90 95Pro Ala Gly His Phe Ala Glu Gly Ser Ser Val Met
Leu Gly Gly Glu 100 105 110Glu Val Asp Gly Val Val Thr Met Trp Gly
Arg Tyr Leu Thr Trp Ala 115 120 125Leu Ser Thr Pro Met Ile Leu Leu
Ala Leu Gly Leu Leu Ala Gly Ser 130 135 140Asn Ala Thr Lys Leu Phe
Thr Ala Ile Thr Phe Asp Ile Ala Met Cys145 150 155 160Val Thr Gly
Leu Ala Ala Ala Leu Thr Thr Ser Ser His Leu Met Arg 165 170 175Trp
Phe Trp Tyr Ala Ile Ser Cys Ala Cys Phe Leu Val Val Leu Tyr 180 185
190Ile Leu Leu Val Glu Trp Ala Gln Asp Ala Lys Ala Ala Gly Thr Ala
195 200 205Asp Met Phe Asn Thr Leu Lys Leu Leu Thr Val Val Met Trp
Leu Gly 210 215 220Tyr Pro Ile Val Trp Ala Leu Gly Val Glu Gly Ile
Ala Val Leu Pro225 230 235 240Val Gly Val Thr Ser Trp Gly Tyr Ser
Phe Leu Asp Ile Val Ala Lys 245 250 255Tyr Ile Phe Ala Phe Leu Leu
Leu Asn Tyr Leu Thr Ser Asn Glu Ser 260 265 270Val Val Ser Gly Ser
Ile Leu Asp Val Pro Ser Ala Ser Gly Thr Pro 275 280 285Ala Asp Asp
29035320PRTArtificial sequenceSynthetic polypeptide 35Met Thr Glu
Thr Leu Pro Pro Val Thr Glu Ser Ala Val Ala Leu Gln1 5 10 15Ala Glu
Val Thr Gln Arg Glu Leu Phe Glu Phe Val Leu Asn Asp Pro 20 25 30Leu
Leu Ala Ser Ser Leu Tyr Ile Asn Ile Ala Leu Ala Gly Leu Ser 35 40
45Ile Leu Leu Phe Val Phe Met Thr Arg Gly Leu Asp Asp Pro Arg Ala
50 55 60Lys Leu Ile Ala Val Ser Thr Ile Leu Val Pro Val Val Ser Ile
Ala65 70 75 80Ser Tyr Thr Gly Leu Ala Ser Gly Leu Thr Ile Ser Val
Leu Glu Met 85 90 95Pro Ala Gly His Phe Ala Glu Gly Ser Ser Val Met
Leu Gly Gly Glu 100 105 110Glu Val Asp Gly Val Val Thr Met Trp Gly
Arg Tyr Leu Thr Trp Ala 115 120 125Leu Ser Thr Pro Met Ile Leu Leu
Ala Leu Gly Leu Leu Ala Gly Ser 130 135 140Asn Ala Thr Lys Leu Phe
Thr Ala Ile Thr Phe Asp Ile Ala Met Cys145 150 155 160Val Thr Gly
Leu Ala Ala Ala Leu Thr Thr Ser Ser His Leu Met Arg 165 170 175Trp
Phe Trp Tyr Ala Ile Ser Cys Ala Cys Phe Leu Val Val Leu Tyr 180 185
190Ile Leu Leu Val Glu Trp Ala Gln Asp Ala Lys Ala Ala Gly Thr Ala
195 200 205Asp Met Phe Asn Thr Leu Lys Leu Leu Thr Val Val Met Trp
Leu Gly 210 215 220Tyr Pro Ile Val Trp Ala Leu Gly Val Glu Gly Ile
Ala Val Leu Pro225 230 235 240Val Gly Val Thr Ser Trp Gly Tyr Ser
Phe Leu Asp Ile Val Ala Lys 245 250 255Tyr Ile Phe Ala Phe Leu Leu
Leu Asn Tyr Leu Thr Ser Asn Glu Ser 260 265 270Val Val Ser Gly Ser
Ile Leu Asp Val Pro Ser Ala Ser Gly Thr Pro 275 280 285Ala Asp Asp
Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr 290 295 300Ile
Pro Leu Asp Gln Ile Asp Ile Asn Phe Cys Tyr Glu Asn Glu Val305 310
315 32036303PRTArtificial sequenceSynthetic polypeptide 36Met Val
Thr Gln Arg Glu Leu Phe Glu Phe Val Leu Asn Asp Pro Leu1 5 10 15Leu
Ala Ser Ser Leu Tyr Ile Asn Ile Ala Leu Ala Gly Leu Ser Ile 20 25
30Leu Leu Phe Val Phe Met Thr Arg Gly Leu Asp Asp Pro Arg Ala Lys
35 40 45Leu Ile Ala Val Ser Thr Ile Leu Val Pro Val Val Ser Ile Ala
Ser 50 55 60Tyr Thr Gly Leu Ala Ser Gly Leu Thr Ile Ser Val Leu Glu
Met Pro65 70 75 80Ala Gly His Phe Ala Glu Gly Ser Ser Val Met Leu
Gly Gly Glu Glu 85 90 95Val Asp Gly Val Val Thr Met Trp Gly Arg Tyr
Leu Thr Trp Ala Leu 100 105 110Ser Thr Pro Met Ile Leu Leu Ala Leu
Gly Leu Leu Ala Gly Ser Asn 115 120 125Ala Thr Lys Leu Phe Thr Ala
Ile Thr Phe Asp Ile Ala Met Cys Val 130 135 140Thr Gly Leu Ala Ala
Ala Leu Thr Thr Ser Ser His Leu Met Arg Trp145 150 155 160Phe Trp
Tyr Ala Ile Ser Cys Ala Cys Phe Leu Val Val Leu Tyr Ile 165 170
175Leu Leu Val Glu Trp Ala Gln Asp Ala Lys Ala Ala Gly Thr Ala Asp
180 185 190Met Phe Asn Thr Leu Lys Leu Leu Thr Val Val Met Trp Leu
Gly Tyr 195 200 205Pro Ile Val Trp Ala Leu Gly Val Glu Gly Ile Ala
Val Leu Pro Val 210 215 220Gly Val Thr Ser Trp Gly Tyr Ser Phe Leu
Asp Ile Val Ala Lys Tyr225 230 235 240Ile Phe Ala Phe Leu Leu Leu
Asn Tyr Leu Thr Ser Asn Glu Ser Val 245 250 255Val Ser Gly Ser Ile
Leu Asp Val Pro Ser Ala Ser Gly Thr Pro Ala 260 265 270Asp Asp Ala
Ala Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile 275 280 285Pro
Leu Asp Gln Ile Asp Ile Asn Phe Cys Tyr Glu Asn Glu Val 290 295
30037365PRTArtificial sequenceSynthetic polypeptide 37Met Arg Arg
Arg Glu Ser Gln Leu Ala Tyr Leu Cys Leu Phe Val Leu1 5 10 15Ile Ala
Gly Trp Ala Pro Arg Leu Thr Glu Ser Ala Pro Asp Leu Ala 20 25 30Glu
Arg Arg Pro Pro Ser Glu Arg Asn Thr Pro Tyr Ala Asn Ile Lys 35 40
45Lys Val Pro Asn Ile Thr Glu Pro Asn Ala Asn Val Gln Leu Asp Gly
50 55 60Trp Ala Leu Tyr Gln Asp Phe Tyr Tyr Leu Ala Gly Ser Asp Lys
Glu65 70 75 80Trp Val Val Gly Pro Ser Asp Gln Cys Tyr Cys Arg Ala
Trp Ser Lys 85 90 95Ser His Gly Thr Asp Arg Glu Gly Glu Ala Ala Val
Val Trp Ala Tyr 100 105 110Ile Val Phe Ala Ile Cys Ile Val Gln Leu
Val Tyr Phe Met Phe Ala 115 120 125Ala Trp Lys Ala Thr Val Gly Trp
Glu Glu Val Tyr Val Asn Ile Ile 130 135 140Glu Leu Val His Ile Ala
Leu Val Ile Trp Val Glu Phe Asp Lys Pro145 150 155 160Ala Met Leu
Tyr Leu Asn Asp Gly Gln Met Val Pro Trp Leu Arg Tyr 165 170 175Ser
Ala Trp Leu Leu Ser Cys Pro Val Ile Leu Ile His Leu Ser Asn 180 185
190Leu Thr Gly Leu Lys Gly Asp Tyr Ser Lys Arg Thr Met Gly Leu Leu
195 200 205Val Ser Asp Ile Gly Thr Ile Val Phe Gly Thr Ser Ala Ala
Leu Ala 210 215 220Pro Pro Asn His Val Lys Val Ile Leu Phe Thr Ile
Gly Leu Leu Tyr225 230 235 240Gly Leu Phe Thr Phe Phe Thr Ala Ala
Lys Val Tyr Ile Glu Ala Tyr 245 250 255His Thr Val Pro Lys Gly Gln
Cys Arg Asn Leu Val Arg Ala Met Ala 260 265 270Trp Thr Tyr Phe Val
Ser Trp Ala Met Phe Pro Ile Leu Phe Ile Leu 275 280 285Gly Arg Glu
Gly Phe Gly His Ile Thr Tyr Phe Gly Ser Ser Ile Gly 290 295 300His
Phe Ile Leu Glu Ile Phe Ser Lys Asn Leu Trp Ser Leu Leu Gly305 310
315 320His Gly Leu Arg Tyr Arg Ile Arg Gln His Ile Ile Ile His Gly
Asn 325 330 335Leu Thr Lys Lys Asn Lys Ile Asn Ile Ala Gly Asp Asn
Val Glu Val 340 345 350Glu Glu Tyr Val Asp Ser Asn Asp Lys Asp Ser
Asp Val 355 360 36538395PRTArtificial sequenceSynthetic polypeptide
38Met Arg Arg Arg Glu Ser Gln Leu Ala Tyr Leu Cys Leu Phe Val Leu1
5 10 15Ile Ala Gly Trp Ala Pro Arg Leu Thr Glu Ser Ala Pro Asp Leu
Ala 20 25 30Glu Arg Arg Pro Pro Ser Glu Arg Asn Thr Pro Tyr Ala Asn
Ile Lys 35 40 45Lys Val Pro Asn Ile Thr Glu Pro Asn Ala Asn Val Gln
Leu Asp Gly 50 55 60Trp Ala Leu Tyr Gln Asp Phe Tyr Tyr Leu Ala Gly
Ser Asp Lys Glu65 70 75 80Trp Val Val Gly Pro Ser Asp Gln Cys Tyr
Cys Arg Ala Trp Ser Lys 85 90 95Ser His Gly Thr Asp Arg Glu Gly Glu
Ala Ala Val Val Trp Ala Tyr 100 105 110Ile Val Phe Ala Ile Cys Ile
Val Gln Leu Val Tyr Phe Met Phe Ala 115 120 125Ala Trp Lys Ala Thr
Val Gly Trp Glu Glu Val Tyr Val Asn Ile Ile 130 135 140Glu Leu Val
His Ile Ala Leu Val Ile Trp Val Glu Phe Asp Lys Pro145 150 155
160Ala Met Leu Tyr Leu Asn Asp Gly Gln Met Val Pro Trp Leu Arg Tyr
165 170 175Ser Ala Trp Leu Leu Ser Cys Pro Val Ile Leu Ile His Leu
Ser Asn 180 185 190Leu Thr Gly Leu Lys Gly Asp Tyr Ser Lys Arg Thr
Met Gly Leu Leu 195 200 205Val Ser Asp Ile Gly Thr Ile Val Phe Gly
Thr Ser Ala Ala Leu Ala 210 215 220Pro Pro Asn His Val Lys Val Ile
Leu Phe Thr Ile Gly Leu Leu Tyr225 230 235 240Gly Leu Phe Thr Phe
Phe Thr Ala Ala Lys Val Tyr Ile Glu Ala Tyr 245 250 255His Thr Val
Pro Lys Gly Gln Cys Arg Asn Leu Val Arg Ala Met Ala 260 265 270Trp
Thr Tyr Phe Val Ser Trp Ala Met Phe Pro Ile Leu Phe Ile Leu 275 280
285Gly Arg Glu Gly Phe Gly His Ile Thr Tyr Phe Gly Ser Ser Ile Gly
290 295 300His Phe Ile Leu Glu Ile Phe Ser Lys Asn Leu Trp Ser Leu
Leu Gly305 310 315 320His Gly Leu Arg Tyr Arg Ile Arg Gln His Ile
Ile Ile His Gly Asn 325 330 335Leu Thr Lys Lys Asn Lys Ile Asn Ile
Ala Gly Asp Asn Val Glu Val 340 345 350Glu Glu Tyr Val Asp Ser Asn
Asp Lys Asp Ser Asp Val Ala Ala Ala 355 360 365Lys Ser Arg Ile Thr
Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile 370 375 380Asp Ile Asn
Val Phe Cys Tyr Glu Asn Glu Val385 390 39539348PRTArtificial
sequenceSynthetic polypeptide 39Met Ser Arg Arg Pro Trp Leu Leu Ala
Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala Gly Ser Ala Gly Ala Ser
Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val Ala Thr Gln Asp Gly Pro
Asp Tyr Val Phe His Arg Ala His Glu 35 40 45Arg Met Leu Phe Gln Thr
Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val 50 55 60Ile Cys Ile Pro Asn
Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu Lys65 70 75 80Ser Asn Gly
Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile Leu Gln Trp 85 90 95Ile Ser
Phe Ala Leu Ser Ala Leu Cys Leu Met Phe Tyr Gly Tyr Gln 100 105
110Thr Trp Lys Ser Thr Cys Gly Trp Glu Glu Ile Tyr Val Ala Thr
Ile
115 120 125Ser Met Ile Lys Phe Ile Ile Glu Tyr Phe His Ser Phe Asp
Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser Asn Gly Asn Lys Thr Lys
Trp Leu Arg Tyr145 150 155 160Ala Ser Trp Leu Leu Thr Cys Pro Val
Ile Leu Ile Arg Leu Ser Asn 165 170 175Leu Thr Gly Leu Ala Asn Asp
Tyr Asn Lys Arg Thr Met Gly Leu Leu 180 185 190Val Ser Asp Ile Gly
Thr Ile Val Trp Gly Thr Thr Ala Ala Leu Ser 195 200 205Lys Gly Tyr
Val Arg Val Ile Phe Phe Leu Met Gly Leu Cys Tyr Gly 210 215 220Ile
Tyr Thr Phe Phe Asn Ala Ala Lys Val Tyr Ile Glu Ala Tyr His225 230
235 240Thr Val Pro Lys Gly Arg Cys Arg Gln Val Val Thr Gly Met Ala
Trp 245 250 255Leu Phe Phe Val Ser Trp Gly Met Phe Pro Ile Leu Phe
Ile Leu Gly 260 265 270Pro Glu Gly Phe Gly Val Leu Ser Lys Tyr Gly
Ser Asn Val Gly His 275 280 285Thr Ile Ile Asp Leu Met Ser Lys Gln
Cys Trp Gly Leu Leu Gly His 290 295 300Tyr Leu Arg Val Leu Ile His
Glu His Ile Leu Ile His Gly Asp Ile305 310 315 320Arg Lys Thr Thr
Lys Leu Asn Ile Gly Gly Thr Glu Ile Glu Val Glu 325 330 335Thr Leu
Val Glu Asp Glu Ala Glu Ala Gly Ala Val 340 34540378PRTArtificial
sequenceSynthetic polypeptide 40Met Ser Arg Arg Pro Trp Leu Leu Ala
Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala Gly Ser Ala Gly Ala Ser
Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val Ala Thr Gln Asp Gly Pro
Asp Tyr Val Phe His Arg Ala His Glu 35 40 45Arg Met Leu Phe Gln Thr
Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val 50 55 60Ile Cys Ile Pro Asn
Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu Lys65 70 75 80Ser Asn Gly
Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile Leu Gln Trp 85 90 95Ile Ser
Phe Ala Leu Ser Ala Leu Cys Leu Met Phe Tyr Gly Tyr Gln 100 105
110Thr Trp Lys Ser Thr Cys Gly Trp Glu Glu Ile Tyr Val Ala Thr Ile
115 120 125Ser Met Ile Lys Phe Ile Ile Glu Tyr Phe His Ser Phe Asp
Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser Asn Gly Asn Lys Thr Lys
Trp Leu Arg Tyr145 150 155 160Ala Ser Trp Leu Leu Thr Cys Pro Val
Ile Leu Ile Arg Leu Ser Asn 165 170 175Leu Thr Gly Leu Ala Asn Asp
Tyr Asn Lys Arg Thr Met Gly Leu Leu 180 185 190Val Ser Asp Ile Gly
Thr Ile Val Trp Gly Thr Thr Ala Ala Leu Ser 195 200 205Lys Gly Tyr
Val Arg Val Ile Phe Phe Leu Met Gly Leu Cys Tyr Gly 210 215 220Ile
Tyr Thr Phe Phe Asn Ala Ala Lys Val Tyr Ile Glu Ala Tyr His225 230
235 240Thr Val Pro Lys Gly Arg Cys Arg Gln Val Val Thr Gly Met Ala
Trp 245 250 255Leu Phe Phe Val Ser Trp Gly Met Phe Pro Ile Leu Phe
Ile Leu Gly 260 265 270Pro Glu Gly Phe Gly Val Leu Ser Lys Tyr Gly
Ser Asn Val Gly His 275 280 285Thr Ile Ile Asp Leu Met Ser Lys Gln
Cys Trp Gly Leu Leu Gly His 290 295 300Tyr Leu Arg Val Leu Ile His
Glu His Ile Leu Ile His Gly Asp Ile305 310 315 320Arg Lys Thr Thr
Lys Leu Asn Ile Gly Gly Thr Glu Ile Glu Val Glu 325 330 335Thr Leu
Val Glu Asp Glu Ala Glu Ala Gly Ala Val Ala Ala Ala Lys 340 345
350Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile Asp
355 360 365Ile Asn Val Phe Cys Tyr Glu Asn Glu Val 370
37541348PRTArtificial sequenceSynthetic
polypeptidemisc_feature(167)..(167)Xaa can be any naturally
occurring amino acid 41Met Ser Arg Arg Pro Trp Leu Leu Ala Leu Ala
Leu Ala Val Ala Leu1 5 10 15Ala Ala Gly Ser Ala Gly Ala Ser Thr Gly
Ser Asp Ala Thr Val Pro 20 25 30Val Ala Thr Gln Asp Gly Pro Asp Tyr
Val Phe His Arg Ala His Glu 35 40 45Arg Met Leu Phe Gln Thr Ser Tyr
Thr Leu Glu Asn Asn Gly Ser Val 50 55 60Ile Cys Ile Pro Asn Asn Gly
Gln Cys Phe Cys Leu Ala Trp Leu Lys65 70 75 80Ser Asn Gly Thr Asn
Ala Glu Lys Leu Ala Ala Asn Ile Leu Gln Trp 85 90 95Ile Ser Phe Ala
Leu Ser Ala Leu Cys Leu Met Phe Tyr Gly Tyr Gln 100 105 110Thr Trp
Lys Ser Thr Cys Gly Trp Glu Glu Ile Tyr Val Ala Thr Ile 115 120
125Ser Met Ile Lys Phe Ile Ile Glu Tyr Phe His Ser Phe Asp Glu Pro
130 135 140Ala Val Ile Tyr Ser Ser Asn Gly Asn Lys Thr Lys Trp Leu
Arg Tyr145 150 155 160Ala Ser Trp Leu Leu Thr Xaa Pro Val Ile Leu
Ile Arg Leu Ser Asn 165 170 175Leu Thr Gly Leu Ala Asn Asp Tyr Asn
Lys Arg Thr Met Gly Leu Leu 180 185 190Val Ser Asp Ile Gly Thr Ile
Val Trp Gly Thr Thr Ala Ala Leu Ser 195 200 205Lys Gly Tyr Val Arg
Val Ile Phe Phe Leu Met Gly Leu Cys Tyr Gly 210 215 220Ile Tyr Thr
Phe Phe Asn Ala Ala Lys Val Tyr Ile Glu Ala Tyr His225 230 235
240Thr Val Pro Lys Gly Arg Cys Arg Gln Val Val Thr Gly Met Ala Trp
245 250 255Leu Phe Phe Val Ser Trp Gly Met Phe Pro Ile Leu Phe Ile
Leu Gly 260 265 270Pro Glu Gly Phe Gly Val Leu Ser Lys Tyr Gly Ser
Asn Val Gly His 275 280 285Thr Ile Ile Asp Leu Met Ser Lys Gln Cys
Trp Gly Leu Leu Gly His 290 295 300Tyr Leu Arg Val Leu Ile His Glu
His Ile Leu Ile His Gly Asp Ile305 310 315 320Arg Lys Thr Thr Lys
Leu Asn Ile Gly Gly Thr Glu Ile Glu Val Glu 325 330 335Thr Leu Val
Glu Asp Glu Ala Glu Ala Gly Ala Val 340 34542378PRTArtificial
sequenceSynthetic polypeptideMISC_FEATURE(167)..(167)Xaa is any
amino acid. 42Met Ser Arg Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala
Val Ala Leu1 5 10 15Ala Ala Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp
Ala Thr Val Pro 20 25 30Val Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe
His Arg Ala His Glu 35 40 45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu
Glu Asn Asn Gly Ser Val 50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys
Phe Cys Leu Ala Trp Leu Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu
Lys Leu Ala Ala Asn Ile Leu Gln Trp 85 90 95Ile Ser Phe Ala Leu Ser
Ala Leu Cys Leu Met Phe Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser
Thr Cys Gly Trp Glu Glu Ile Tyr Val Ala Thr Ile 115 120 125Ser Met
Ile Lys Phe Ile Ile Glu Tyr Phe His Ser Phe Asp Glu Pro 130 135
140Ala Val Ile Tyr Ser Ser Asn Gly Asn Lys Thr Lys Trp Leu Arg
Tyr145 150 155 160Ala Ser Trp Leu Leu Thr Xaa Pro Val Ile Leu Ile
Arg Leu Ser Asn 165 170 175Leu Thr Gly Leu Ala Asn Asp Tyr Asn Lys
Arg Thr Met Gly Leu Leu 180 185 190Val Ser Asp Ile Gly Thr Ile Val
Trp Gly Thr Thr Ala Ala Leu Ser 195 200 205Lys Gly Tyr Val Arg Val
Ile Phe Phe Leu Met Gly Leu Cys Tyr Gly 210 215 220Ile Tyr Thr Phe
Phe Asn Ala Ala Lys Val Tyr Ile Glu Ala Tyr His225 230 235 240Thr
Val Pro Lys Gly Arg Cys Arg Gln Val Val Thr Gly Met Ala Trp 245 250
255Leu Phe Phe Val Ser Trp Gly Met Phe Pro Ile Leu Phe Ile Leu Gly
260 265 270Pro Glu Gly Phe Gly Val Leu Ser Lys Tyr Gly Ser Asn Val
Gly His 275 280 285Thr Ile Ile Asp Leu Met Ser Lys Gln Cys Trp Gly
Leu Leu Gly His 290 295 300Tyr Leu Arg Val Leu Ile His Glu His Ile
Leu Ile His Gly Asp Ile305 310 315 320Arg Lys Thr Thr Lys Leu Asn
Ile Gly Gly Thr Glu Ile Glu Val Glu 325 330 335Thr Leu Val Glu Asp
Glu Ala Glu Ala Gly Ala Val Ala Ala Ala Lys 340 345 350Ser Arg Ile
Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile Asp 355 360 365Ile
Asn Val Phe Cys Tyr Glu Asn Glu Val 370 37543309PRTArtificial
sequenceSynthetic polypeptide 43Met Asp Tyr Gly Gly Ala Leu Ser Ala
Val Gly Leu Phe Gln Thr Ser1 5 10 15Tyr Thr Leu Glu Asn Asn Gly Ser
Val Ile Cys Ile Pro Asn Asn Gly 20 25 30Gln Cys Phe Cys Leu Ala Trp
Leu Lys Ser Asn Gly Thr Asn Ala Glu 35 40 45Lys Leu Ala Ala Asn Ile
Leu Gln Trp Ile Ser Phe Ala Leu Ser Ala 50 55 60Leu Cys Leu Met Phe
Tyr Gly Tyr Gln Thr Trp Lys Ser Thr Cys Gly65 70 75 80Trp Glu Glu
Ile Tyr Val Ala Thr Ile Ser Met Ile Lys Phe Ile Ile 85 90 95Glu Tyr
Phe His Ser Phe Asp Glu Pro Ala Val Ile Tyr Ser Ser Asn 100 105
110Gly Asn Lys Thr Lys Trp Leu Arg Tyr Ala Ser Trp Leu Leu Thr Cys
115 120 125Pro Val Ile Leu Ile Arg Leu Ser Asn Leu Thr Gly Leu Ala
Asn Asp 130 135 140Tyr Asn Lys Arg Thr Met Gly Leu Leu Val Ser Asp
Ile Gly Thr Ile145 150 155 160Val Trp Gly Thr Thr Ala Ala Leu Ser
Lys Gly Tyr Val Arg Val Ile 165 170 175Phe Phe Leu Met Gly Leu Cys
Tyr Gly Ile Tyr Thr Phe Phe Asn Ala 180 185 190Ala Lys Val Tyr Ile
Glu Ala 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 Lys Tyr Gly Ser Asn Val Gly His Thr Ile Ile Asp Leu Met
Ser 245 250 255Lys Gln 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
Val30544339PRTArtificial sequenceSynthetic polypeptide 44Met Asp
Tyr Gly Gly Ala Leu Ser Ala Val Gly Leu Phe Gln Thr Ser1 5 10 15Tyr
Thr Leu Glu Asn Asn Gly Ser Val Ile Cys Ile Pro Asn Asn Gly 20 25
30Gln Cys Phe Cys Leu Ala Trp Leu Lys Ser Asn Gly Thr Asn Ala Glu
35 40 45Lys Leu Ala Ala Asn Ile Leu Gln Trp Ile Ser Phe Ala Leu Ser
Ala 50 55 60Leu Cys Leu Met Phe Tyr Gly Tyr Gln Thr Trp Lys Ser Thr
Cys Gly65 70 75 80Trp Glu Glu Ile Tyr Val Ala Thr Ile Ser Met Ile
Lys Phe Ile Ile 85 90 95Glu Tyr Phe His Ser Phe Asp Glu Pro Ala Val
Ile Tyr Ser Ser Asn 100 105 110Gly Asn Lys Thr Lys Trp Leu Arg Tyr
Ala Ser Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Leu Ile Arg Leu
Ser Asn Leu Thr Gly Leu Ala Asn Asp 130 135 140Tyr Asn Lys Arg Thr
Met Gly Leu Leu Val Ser Asp Ile Gly Thr Ile145 150 155 160Val Trp
Gly Thr Thr Ala Ala Leu Ser Lys Gly Tyr Val Arg Val Ile 165 170
175Phe Phe Leu Met Gly Leu Cys Tyr Gly Ile Tyr Thr Phe Phe Asn Ala
180 185 190Ala Lys Val Tyr Ile Glu Ala 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 Lys Tyr Gly Ser Asn Val
Gly His Thr Ile Ile Asp Leu Met Ser 245 250 255Lys Gln 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 Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu
Gly305 310 315 320Glu Tyr Ile Pro Leu Asp Gln Ile Asp Ile Asn Val
Phe Cys Tyr Glu 325 330 335Asn Glu Val45310PRTArtificial
sequenceSynthetic polypeptide 45Met 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 Ser 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 Ser Met Val Lys Val Ile Leu 85 90 95Glu Phe
Phe Phe Ser Phe Lys Asn Pro Ser Met Leu Tyr Leu Ala Thr 100 105
110Gly His Arg Val Lys Trp Leu Arg Tyr Ala Ser Trp Leu Leu Thr Cys
115 120 125Pro Val Ile Leu Ile Arg 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 Lys Tyr Gly Ser Asn Val Gly His Thr Ile Ile Asp Leu Met
Ser 245 250 255Lys Gln 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 Pro305
31046340PRTArtificial sequenceSynthetic polypeptide 46Met 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 Ser 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 Ser Met Val Lys
Val Ile Leu 85 90 95Glu Phe Phe Phe Ser Phe Lys Asn Pro Ser Met Leu
Tyr Leu Ala Thr 100 105 110Gly His Arg Val Lys Trp Leu Arg Tyr Ala
Ser Trp Leu Leu Thr Cys 115
120 125Pro Val Ile Leu Ile Arg 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 Lys Tyr Gly Ser Asn Val Gly His Thr Ile Ile Asp Leu Met Ser
245 250 255Lys Gln 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 Pro Ala Ala
Ala Lys Ser Arg Ile Thr Ser Glu305 310 315 320Gly Glu Tyr Ile Pro
Leu Asp Gln Ile Asp Ile Asn Val Phe Cys Tyr 325 330 335Glu Asn Glu
Val 34047344PRTArtificial sequenceSynthetic polypeptide 47Met Ser
Arg Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala
Ala Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro 20 25
30Val Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His Glu
35 40 45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser
Val 50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp
Leu Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn
Ile Leu Gln Trp 85 90 95Ile Ser Phe Ala Leu Ser Ala Leu Cys Leu Met
Phe Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp Glu
Glu Ile Tyr Val Ala Thr Ile 115 120 125Ser Met Ile Lys Phe Ile Ile
Glu Tyr Phe His Ser Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr Ser
Ser Asn Gly Asn Lys Thr Lys Trp Leu Arg Tyr145 150 155 160Ala Ser
Trp Leu Leu Thr Cys Pro Val Leu Leu Ile Arg Leu Ser Asn 165 170
175Leu Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr Met Gly Leu Leu
180 185 190Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr Ser Ala
Met Cys 195 200 205Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser
Leu Ser Tyr Gly 210 215 220Met Tyr Thr Tyr Phe His Ala Ala Lys Val
Tyr Ile Glu Ala Phe His225 230 235 240Thr Val Pro Lys Gly Ile Cys
Arg Glu Leu Val Arg Val Met Ala Trp 245 250 255Thr Phe Phe Val Ala
Trp Gly Met Phe Pro Val Leu Phe Leu Leu Gly 260 265 270Thr Glu Gly
Phe Gly His Ile Ser Lys Tyr Gly Ser Asn Ile Gly His 275 280 285Ser
Ile Leu Asp Leu Ile Ala Lys Gln Met Trp Gly Val Leu Gly Asn 290 295
300Tyr Leu Arg Val Lys Ile His Glu His Ile Leu Leu Tyr Gly Asp
Ile305 310 315 320Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu
Met Glu Val Glu 325 330 335Thr Leu Val Ala Glu Glu Glu Asp
34048374PRTArtificial sequenceSynthetic polypeptide 48Met Ser Arg
Arg Pro Trp Leu Leu Ala Leu Ala Leu Ala Val Ala Leu1 5 10 15Ala Ala
Gly Ser Ala Gly Ala Ser Thr Gly Ser Asp Ala Thr Val Pro 20 25 30Val
Ala Thr Gln Asp Gly Pro Asp Tyr Val Phe His Arg Ala His Glu 35 40
45Arg Met Leu Phe Gln Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser Val
50 55 60Ile Cys Ile Pro Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu
Lys65 70 75 80Ser Asn Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile
Leu Gln Trp 85 90 95Ile Ser Phe Ala Leu Ser Ala Leu Cys Leu Met Phe
Tyr Gly Tyr Gln 100 105 110Thr Trp Lys Ser Thr Cys Gly Trp Glu Glu
Ile Tyr Val Ala Thr Ile 115 120 125Ser Met Ile Lys Phe Ile Ile Glu
Tyr Phe His Ser Phe Asp Glu Pro 130 135 140Ala Val Ile Tyr Ser Ser
Asn Gly Asn Lys Thr Lys Trp Leu Arg Tyr145 150 155 160Ala Ser Trp
Leu Leu Thr Cys Pro Val Leu Leu Ile Arg Leu Ser Asn 165 170 175Leu
Thr Gly Leu Lys Asp Asp Tyr Ser Lys Arg Thr Met Gly Leu Leu 180 185
190Val Ser Asp Val Gly Cys Ile Val Trp Gly Ala Thr Ser Ala Met Cys
195 200 205Thr Gly Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser
Tyr Gly 210 215 220Met Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile
Glu Ala Phe His225 230 235 240Thr Val Pro Lys Gly Ile Cys Arg Glu
Leu Val Arg Val Met Ala Trp 245 250 255Thr Phe Phe Val Ala Trp Gly
Met Phe Pro Val Leu Phe Leu Leu Gly 260 265 270Thr Glu Gly Phe Gly
His Ile Ser Lys Tyr Gly Ser Asn Ile Gly His 275 280 285Ser Ile Leu
Asp Leu Ile Ala Lys Gln Met Trp Gly Val Leu Gly Asn 290 295 300Tyr
Leu Arg Val Lys Ile His Glu His Ile Leu Leu Tyr Gly Asp Ile305 310
315 320Arg Lys Lys Gln Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val
Glu 325 330 335Thr Leu Val Ala Glu Glu Glu Asp Ala Ala Ala Lys Ser
Arg Ile Thr 340 345 350Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile
Asp Ile Asn Val Phe 355 360 365Cys Tyr Glu Asn Glu Val
37049305PRTArtificial sequenceSynthetic polypeptide 49Met Asp Tyr
Gly Gly Ala Leu Ser Ala Val Gly Leu Phe Gln Thr Ser1 5 10 15Tyr Thr
Leu Glu Asn Asn Gly Ser Val Ile Cys Ile Pro Asn Asn Gly 20 25 30Gln
Cys Phe Cys Leu Ala Trp Leu Lys Ser Asn Gly Thr Asn Ala Glu 35 40
45Lys Leu Ala Ala Asn Ile Leu Gln Trp Ile Ser Phe Ala Leu Ser Ala
50 55 60Leu Cys Leu Met Phe Tyr Gly Tyr Gln Thr Trp Lys Ser Thr Cys
Gly65 70 75 80Trp Glu Glu Ile Tyr Val Ala Thr Ile Ser Met Ile Lys
Phe Ile Ile 85 90 95Glu Tyr Phe His Ser Phe Asp Glu Pro Ala Val Ile
Tyr Ser Ser Asn 100 105 110Gly Asn Lys Thr Lys Trp Leu Arg Tyr Ala
Ser Trp Leu Leu Thr Cys 115 120 125Pro Val Leu Leu Ile Arg Leu Ser
Asn Leu Thr Gly Leu Lys Asp Asp 130 135 140Tyr Ser Lys Arg Thr Met
Gly Leu Leu Val Ser Asp Val Gly Cys Ile145 150 155 160Val Trp Gly
Ala Thr Ser Ala Met Cys Thr Gly Trp Thr Lys Ile Leu 165 170 175Phe
Phe Leu Ile Ser Leu Ser Tyr Gly Met Tyr Thr Tyr Phe His Ala 180 185
190Ala Lys Val Tyr Ile Glu Ala Phe His Thr Val Pro Lys Gly Ile Cys
195 200 205Arg Glu Leu Val Arg Val Met Ala Trp Thr Phe Phe Val Ala
Trp Gly 210 215 220Met Phe Pro Val Leu Phe Leu Leu Gly Thr Glu Gly
Phe Gly His Ile225 230 235 240Ser Lys Tyr Gly Ser Asn Ile Gly His
Ser Ile Leu Asp Leu Ile Ala 245 250 255Lys Gln Met Trp Gly Val Leu
Gly Asn Tyr Leu Arg Val Lys Ile His 260 265 270Glu His Ile Leu Leu
Tyr Gly Asp Ile Arg Lys Lys Gln Lys Ile Thr 275 280 285Ile Ala Gly
Gln Glu Met Glu Val Glu Thr Leu Val Ala Glu Glu Glu 290 295
300Asp30550335PRTArtificial sequenceSynthetic polypeptide 50Met Asp
Tyr Gly Gly Ala Leu Ser Ala Val Gly Leu Phe Gln Thr Ser1 5 10 15Tyr
Thr Leu Glu Asn Asn Gly Ser Val Ile Cys Ile Pro Asn Asn Gly 20 25
30Gln Cys Phe Cys Leu Ala Trp Leu Lys Ser Asn Gly Thr Asn Ala Glu
35 40 45Lys Leu Ala Ala Asn Ile Leu Gln Trp Ile Ser Phe Ala Leu Ser
Ala 50 55 60Leu Cys Leu Met Phe Tyr Gly Tyr Gln Thr Trp Lys Ser Thr
Cys Gly65 70 75 80Trp Glu Glu Ile Tyr Val Ala Thr Ile Ser Met Ile
Lys Phe Ile Ile 85 90 95Glu Tyr Phe His Ser Phe Asp Glu Pro Ala Val
Ile Tyr Ser Ser Asn 100 105 110Gly Asn Lys Thr Lys Trp Leu Arg Tyr
Ala Ser Trp Leu Leu Thr Cys 115 120 125Pro Val Leu Leu Ile Arg Leu
Ser Asn Leu Thr Gly Leu Lys Asp Asp 130 135 140Tyr Ser Lys Arg Thr
Met Gly Leu Leu Val Ser Asp Val Gly Cys Ile145 150 155 160Val Trp
Gly Ala Thr Ser Ala Met Cys Thr Gly Trp Thr Lys Ile Leu 165 170
175Phe Phe Leu Ile Ser Leu Ser Tyr Gly Met Tyr Thr Tyr Phe His Ala
180 185 190Ala Lys Val Tyr Ile Glu Ala Phe His Thr Val Pro Lys Gly
Ile Cys 195 200 205Arg Glu Leu Val Arg Val Met Ala Trp Thr Phe Phe
Val Ala Trp Gly 210 215 220Met Phe Pro Val Leu Phe Leu Leu Gly Thr
Glu Gly Phe Gly His Ile225 230 235 240Ser Lys Tyr Gly Ser Asn Ile
Gly His Ser Ile Leu Asp Leu Ile Ala 245 250 255Lys Gln Met Trp Gly
Val Leu Gly Asn Tyr Leu Arg Val Lys Ile His 260 265 270Glu His Ile
Leu Leu Tyr Gly Asp Ile Arg Lys Lys Gln Lys Ile Thr 275 280 285Ile
Ala Gly Gln Glu Met Glu Val Glu Thr Leu Val Ala Glu Glu Glu 290 295
300Asp Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile
Pro305 310 315 320Leu Asp Gln Ile Asp Ile Asn Val Phe Cys Tyr Glu
Asn Glu Val 325 330 33551350PRTArtificial sequenceSynthetic
polypeptide 51Met Val Ser Arg Arg Pro Trp Leu Leu Ala Leu Ala Leu
Ala Val Ala1 5 10 15Leu Ala Ala Gly Ser Ala Gly Ala Ser Thr Gly Ser
Asp Ala Thr Val 20 25 30Pro Val Ala Thr Gln Asp Gly Pro Asp Tyr Val
Phe His Arg Ala His 35 40 45Glu Arg Met Leu Phe Gln Thr Ser Tyr Thr
Leu Glu Asn Asn Gly Ser 50 55 60Val Ile Cys Ile Pro Asn Asn Gly Gln
Cys Phe Cys Leu Ala Trp Leu65 70 75 80Lys Ser Asn Gly Thr Asn Ala
Glu Lys Leu Ala Ala Asn Ile Leu Gln 85 90 95Trp Val Ser Phe Ala Leu
Ser Val Ala Cys Leu Gly Trp Tyr Ala Tyr 100 105 110Gln Ala Trp Arg
Ala Thr Cys Gly Trp Glu Glu Val Tyr Val Ala Leu 115 120 125Ile Ser
Met Met Lys Ser Ile Ile Glu Ala Phe His Ser Phe Asp Ser 130 135
140Pro Ala Thr Leu Trp Leu Ser Ser Gly Asn Gly Val Lys Trp Met
Arg145 150 155 160Tyr Gly Ser Trp Leu Leu Thr Cys Pro Val Ile Leu
Ile Arg Leu Ser 165 170 175Asn Leu Thr Gly Leu Lys Asp Asp Tyr Ser
Lys Arg Thr Met Gly Leu 180 185 190Leu Val Ser Asp Val Gly Cys Ile
Val Trp Gly Ala Thr Ser Ala Met 195 200 205Cys Thr Gly Trp Thr Lys
Ile Leu Phe Phe Leu Ile Ser Leu Ser Tyr 210 215 220Gly Met Tyr Thr
Tyr Phe His Ala Ala Lys Val Tyr Ile Glu Ala Phe225 230 235 240His
Thr Val Pro Lys Gly Leu Cys Arg Gln Leu Val Arg Ala Met Ala 245 250
255Trp Leu Phe Phe Val Ser Trp Gly Met Phe Pro Val Leu Phe Leu Leu
260 265 270Gly Pro Glu Gly Phe Gly His Ile Ser Lys Tyr Gly Ser Asn
Ile Gly 275 280 285His Ser Ile Leu Asp Leu Ile Ala Lys Gln Met Trp
Gly Val Leu Gly 290 295 300Asn Tyr Leu Arg Val Lys Ile His Glu His
Ile Leu Leu Tyr Gly Asp305 310 315 320Ile Arg Lys Lys Gln Lys Ile
Thr Ile Ala Gly Gln Glu Met Glu Val 325 330 335Glu Thr Leu Val Ala
Glu Glu Glu Asp Lys Tyr Glu Ser Ser 340 345 35052380PRTArtificial
sequenceSynthetic polypeptide 52Met Val Ser Arg Arg Pro Trp Leu Leu
Ala Leu Ala Leu Ala Val Ala1 5 10 15Leu Ala Ala Gly Ser Ala Gly Ala
Ser Thr Gly Ser Asp Ala Thr Val 20 25 30Pro Val Ala Thr Gln Asp Gly
Pro Asp Tyr Val Phe His Arg Ala His 35 40 45Glu Arg Met Leu Phe Gln
Thr Ser Tyr Thr Leu Glu Asn Asn Gly Ser 50 55 60Val Ile Cys Ile Pro
Asn Asn Gly Gln Cys Phe Cys Leu Ala Trp Leu65 70 75 80Lys Ser Asn
Gly Thr Asn Ala Glu Lys Leu Ala Ala Asn Ile Leu Gln 85 90 95Trp Val
Ser Phe Ala Leu Ser Val Ala Cys Leu Gly Trp Tyr Ala Tyr 100 105
110Gln Ala Trp Arg Ala Thr Cys Gly Trp Glu Glu Val Tyr Val Ala Leu
115 120 125Ile Ser Met Met Lys Ser Ile Ile Glu Ala Phe His Ser Phe
Asp Ser 130 135 140Pro Ala Thr Leu Trp Leu Ser Ser Gly Asn Gly Val
Lys Trp Met Arg145 150 155 160Tyr Gly Ser Trp Leu Leu Thr Cys Pro
Val Ile Leu Ile Arg Leu Ser 165 170 175Asn Leu Thr Gly Leu Lys Asp
Asp Tyr Ser Lys Arg Thr Met Gly Leu 180 185 190Leu Val Ser Asp Val
Gly Cys Ile Val Trp Gly Ala Thr Ser Ala Met 195 200 205Cys Thr Gly
Trp Thr Lys Ile Leu Phe Phe Leu Ile Ser Leu Ser Tyr 210 215 220Gly
Met Tyr Thr Tyr Phe His Ala Ala Lys Val Tyr Ile Glu Ala Phe225 230
235 240His Thr Val Pro Lys Gly Leu Cys Arg Gln Leu Val Arg Ala Met
Ala 245 250 255Trp Leu Phe Phe Val Ser Trp Gly Met Phe Pro Val Leu
Phe Leu Leu 260 265 270Gly Pro Glu Gly Phe Gly His Ile Ser Lys Tyr
Gly Ser Asn Ile Gly 275 280 285His Ser Ile Leu Asp Leu Ile Ala Lys
Gln Met Trp Gly Val Leu Gly 290 295 300Asn Tyr Leu Arg Val Lys Ile
His Glu His Ile Leu Leu Tyr Gly Asp305 310 315 320Ile Arg Lys Lys
Gln Lys Ile Thr Ile Ala Gly Gln Glu Met Glu Val 325 330 335Glu Thr
Leu Val Ala Glu Glu Glu Asp Lys Tyr Glu Ser Ser Ala Ala 340 345
350Ala Lys Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln
355 360 365Ile Asp Ile Asn Val Phe Cys Tyr Glu Asn Glu Val 370 375
38053310PRTArtificial sequenceSynthetic polypeptide 53Met Asp Tyr
Gly Gly Ala Leu Ser Ala Val Gly Leu Phe Gln Thr Ser1 5 10 15Tyr Thr
Leu Glu Asn Asn Gly Ser Val Ile Cys Ile Pro Asn Asn Gly 20 25 30Gln
Cys Phe Cys Leu Ala Trp Leu Lys Ser Asn Gly Thr Asn Ala Glu 35 40
45Lys Leu Ala Ala Asn Ile Leu Gln Trp Val Ser Phe Ala Leu Ser Val
50 55 60Ala Cys Leu Gly Trp Tyr Ala Tyr Gln Ala Trp Arg Ala Thr Cys
Gly65 70 75 80Trp Glu Glu Val Tyr Val Ala Leu Ile Ser Met Met Lys
Ser Ile Ile 85 90 95Glu Ala Phe His Ser Phe Asp Ser Pro Ala Thr Leu
Trp Leu Ser Ser 100 105 110Gly Asn Gly Val Lys Trp Met Arg Tyr Gly
Ser Trp Leu Leu Thr Cys
115 120 125Pro Val Ile Leu Ile Arg Leu Ser Asn Leu Thr Gly Leu Lys
Asp Asp 130 135 140Tyr Ser Lys Arg Thr Met Gly Leu Leu Val Ser Asp
Val Gly Cys Ile145 150 155 160Val Trp Gly Ala Thr Ser Ala Met Cys
Thr Gly Trp Thr Lys Ile Leu 165 170 175Phe Phe Leu Ile Ser Leu Ser
Tyr Gly Met Tyr Thr Tyr Phe His Ala 180 185 190Ala Lys Val Tyr Ile
Glu Ala Phe His Thr Val Pro Lys Gly Leu Cys 195 200 205Arg Gln Leu
Val Arg Ala Met Ala Trp Leu Phe Phe Val Ser Trp Gly 210 215 220Met
Phe Pro Val Leu Phe Leu Leu Gly Pro Glu Gly Phe Gly His Ile225 230
235 240Ser Lys Tyr Gly Ser Asn Ile Gly His Ser Ile Leu Asp Leu Ile
Ala 245 250 255Lys Gln Met Trp Gly Val Leu Gly Asn Tyr Leu Arg Val
Lys Ile His 260 265 270Glu His Ile Leu Leu Tyr Gly Asp Ile Arg Lys
Lys Gln Lys Ile Thr 275 280 285Ile Ala Gly Gln Glu Met Glu Val Glu
Thr Leu Val Ala Glu Glu Glu 290 295 300Asp Lys Tyr Glu Ser Ser305
31054340PRTArtificial sequenceSynthetic polypeptide 54Met Asp Tyr
Gly Gly Ala Leu Ser Ala Val Gly Leu Phe Gln Thr Ser1 5 10 15Tyr Thr
Leu Glu Asn Asn Gly Ser Val Ile Cys Ile Pro Asn Asn Gly 20 25 30Gln
Cys Phe Cys Leu Ala Trp Leu Lys Ser Asn Gly Thr Asn Ala Glu 35 40
45Lys Leu Ala Ala Asn Ile Leu Gln Trp Val Ser Phe Ala Leu Ser Val
50 55 60Ala Cys Leu Gly Trp Tyr Ala Tyr Gln Ala Trp Arg Ala Thr Cys
Gly65 70 75 80Trp Glu Glu Val Tyr Val Ala Leu Ile Ser Met Met Lys
Ser Ile Ile 85 90 95Glu Ala Phe His Ser Phe Asp Ser Pro Ala Thr Leu
Trp Leu Ser Ser 100 105 110Gly Asn Gly Val Lys Trp Met Arg Tyr Gly
Ser Trp Leu Leu Thr Cys 115 120 125Pro Val Ile Leu Ile Arg Leu Ser
Asn Leu Thr Gly Leu Lys Asp Asp 130 135 140Tyr Ser Lys Arg Thr Met
Gly Leu Leu Val Ser Asp Val Gly Cys Ile145 150 155 160Val Trp Gly
Ala Thr Ser Ala Met Cys Thr Gly Trp Thr Lys Ile Leu 165 170 175Phe
Phe Leu Ile Ser Leu Ser Tyr Gly Met Tyr Thr Tyr Phe His Ala 180 185
190Ala Lys Val Tyr Ile Glu Ala Phe His Thr Val Pro Lys Gly Leu Cys
195 200 205Arg Gln Leu Val Arg Ala Met Ala Trp Leu Phe Phe Val Ser
Trp Gly 210 215 220Met Phe Pro Val Leu Phe Leu Leu Gly Pro Glu Gly
Phe Gly His Ile225 230 235 240Ser Lys Tyr Gly Ser Asn Ile Gly His
Ser Ile Leu Asp Leu Ile Ala 245 250 255Lys Gln Met Trp Gly Val Leu
Gly Asn Tyr Leu Arg Val Lys Ile His 260 265 270Glu His Ile Leu Leu
Tyr Gly Asp Ile Arg Lys Lys Gln Lys Ile Thr 275 280 285Ile Ala Gly
Gln Glu Met Glu Val Glu Thr Leu Val Ala Glu Glu Glu 290 295 300Asp
Lys Tyr Glu Ser Ser Ala Ala Ala Lys Ser Arg Ile Thr Ser Glu305 310
315 320Gly Glu Tyr Ile Pro Leu Asp Gln Ile Asp Ile Asn Val Phe Cys
Tyr 325 330 335Glu Asn Glu Val 3405511PRTArtificial
sequenceSynthetic polypeptide 55Met Asp Tyr Gly Gly Ala Leu Ser Ala
Val Gly1 5 105620PRTArtificial sequenceSynthetic polypeptide 56Lys
Ser Arg Ile Thr Ser Glu Gly Glu Tyr Ile Pro Leu Asp Gln Ile1 5 10
15Asp Ile Asn Val 20575PRTArtificial sequenceSynthetic polypeptide
57Val Lys Glu Ser Leu1 5585PRTArtificial sequenceSynthetic
polypeptide 58Val Leu Gly Ser Leu1 55916PRTArtificial
sequenceSynthetic polypeptide 59Asn Ala Asn Ser Phe Cys Tyr Glu Asn
Glu Val Ala Leu Thr Ser Lys1 5 10 15606PRTArtificial
sequenceSynthetic polypeptideMISC_FEATURE(2)..(2)Xaa is any amino
acid. 60Phe Xaa Tyr Glu Asn Glu1 5617PRTArtificial
sequenceSynthetic polypeptide 61Phe Cys Tyr Glu Asn Glu Val1
56226PRTArtificial sequenceSynthetic polypeptide 62Met 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 20 256327PRTArtificial
sequenceSynthetic polypeptide 63Met Ala Gly His Ser Asn Ser Met Ala
Leu Phe Ser Phe Ser Leu Leu1 5 10 15Trp Leu Cys Ser Gly Val Leu Gly
Thr Glu Phe 20 256423PRTArtificial sequenceSynthetic polypeptide
64Met Gly Leu Arg Ala Leu Met Leu Trp Leu Leu Ala Ala Ala Gly Leu1
5 10 15Val Arg Glu Ser Leu Gln Gly 206518PRTArtificial
sequenceSynthetic polypeptide 65Met Arg Gly Thr Pro Leu Leu Leu Val
Val Ser Leu Phe Ser Leu Leu1 5 10 15Gln Asp6618PRTArtificial
sequenceSynthetic polypeptide 66Met Thr Glu Thr Leu Pro Pro Val Thr
Glu Ser Ala Val Ala Leu Gln1 5 10 15Ala Glu
* * * * *