U.S. patent application number 14/586573 was filed with the patent office on 2015-08-27 for designer ligands of tgf-beta superfamily.
The applicant listed for this patent is Joint Center for Biosciences, The Salk Institute for Biological Studies. Invention is credited to George Allendorph, Senyon Choe, Mike Isaacs.
Application Number | 20150239950 14/586573 |
Document ID | / |
Family ID | 42666199 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150239950 |
Kind Code |
A1 |
Choe; Senyon ; et
al. |
August 27, 2015 |
DESIGNER LIGANDS OF TGF-BETA SUPERFAMILY
Abstract
The present disclosure relates to chimeric polypeptide having
TGF-beta activity, nucleic acids encoding the polypeptides, and
host cells for producing the polypeptides.
Inventors: |
Choe; Senyon; (Solana Beach,
CA) ; Allendorph; George; (San Diego, CA) ;
Isaacs; Mike; (Redwood City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Salk Institute for Biological Studies
Joint Center for Biosciences |
La Jolla
Incheon |
CA |
US
KR |
|
|
Family ID: |
42666199 |
Appl. No.: |
14/586573 |
Filed: |
December 30, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12712069 |
Feb 24, 2010 |
8952130 |
|
|
14586573 |
|
|
|
|
61155066 |
Feb 24, 2009 |
|
|
|
Current U.S.
Class: |
514/8.9 ;
435/375; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 5/00 20180101; A61P
1/02 20180101; A61P 11/00 20180101; A61P 35/00 20180101; A61P 19/00
20180101; A61P 25/16 20180101; A61P 5/16 20180101; A61P 21/00
20180101; A61P 37/06 20180101; A61P 19/10 20180101; A61P 31/04
20180101; A61P 5/24 20180101; A61K 38/00 20130101; A61P 25/14
20180101; A61P 13/12 20180101; A61P 37/08 20180101; A61P 43/00
20180101; A61P 25/28 20180101; A61P 5/18 20180101; A61P 29/00
20180101; A61P 21/02 20180101; C07K 14/495 20130101; A61P 3/04
20180101 |
International
Class: |
C07K 14/495 20060101
C07K014/495 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] The invention was funded, in part, by a grant from the
National Institutes of Health grant number HD013527. The government
has certain right in this invention.
Claims
1. A recombinant TGF-.beta. ligand polypeptide having at least 95%
sequence identity to an amino acid sequence comprising a first
segment of amino acid residues comprising amino acid residues 1 to
32 of SEQ ID NO:2 ("1b"); a second segment of amino acid residues
comprising amino acid residues 33 to x.sub.16 of SEQ ID NO:2
("1b"), wherein: x.sub.16 is residue 45, 46, 47, of 48 of SEQ ID
NO: 2; a third segment of amino acid residues comprising amino acid
residues x.sub.17 to x.sub.1 of SEQ ID NO:2 ("3b"), or amino acid
residues x.sub.2 to x.sub.3 of SEQ ID NO:5 ("3a"), wherein:
x.sub.17 is x.sub.16+1 of SEQ ID NO: 2, x.sub.1 is residue 65, 66,
67, or 68 of SEQ ID NO:2, x.sub.2 is residue 42, 43, 44, or 45 of
SEQ ID NO:5, and x.sub.3 is residue 61, 62, 63, or 64 of SEQ ID
NO:5; and a fourth segment of amino acid residues comprising amino
acid residues x.sub.4 to x.sub.5 of SEQ ID NO:5 ("4a") or amino
acid residues x.sub.6 to x.sub.7 of SEQ ID NO:2 ("4b"), wherein:
x.sub.4 is x.sub.1 of SEQ ID NO: 5 if the third segment comprises
3b, or x.sub.4 is x.sub.3+1 of SEQ ID NO: 5 if the third segment
comprises 3a, x.sub.5 is residue 78, 79, 80, 81, 82, 83 or 84 of
SEQ ID NO:5, x.sub.6 is x.sub.3 of SEQ ID NO: 2 if the third
segment comprises 3a, or x.sub.6 is x.sub.1+1 of SEQ ID NO: 2 if
the third segment comprises 3b, x.sub.7 is residue 76, 77, 78, 79,
80, 81 or 82 of SEQ ID NO:2, and; a fifth segment of amino acid
residues comprising amino acid residues x.sub.8 to x.sub.9 of SEQ
ID NO:5 ("5a") or amino acid residues x.sub.10 to x.sub.11 of SEQ
ID NO:2 ("5b"), wherein: x.sub.8 is x.sub.5 of SEQ ID NO: 5 if the
fourth segment comprises 4b, or x.sub.8 is x.sub.5+1 of SEQ ID NO:
5 if the fourth segment comprises 4a, x.sub.9 is residue 90, 91,
92, 93, 94, 95 or 96 of SEQ ID NO:5, x.sub.10 is x.sub.7 of SEQ ID
NO: 2 if the fourth segment comprises 4a, or x.sub.10 is x.sub.7+1
of SEQ ID NO: 2 if the fourth segment comprises 4b, x.sub.11 is
residue 88, 89, 90, 91, 92, 93, or 94 of SEQ ID NO:2, and a sixth
segment comprising amino acid residues x.sub.12 to x.sub.13 of SEQ
ID NO:5 ("6a") or amino acid residues x.sub.14 to x.sub.15 of SEQ
ID NO:2 ("6b"), wherein: x.sub.12 is x.sub.9 of SEQ ID NO: 5 if the
fifth segment comprises 5b, or x.sub.12 is x.sub.9+1 of SEQ ID NO:
5 if the fifth segment comprises 5a, x.sub.13 is residue 114, 115,
or 116 of SEQ ID NO:5, x.sub.14 is x.sub.11 of SEQ ID NO: 2 if the
fifth segment comprises 5a, or x.sub.14 is x.sub.11+1 of SEQ ID NO:
2 if the fifth segment comprises 5b, x.sub.15 is residue 112, 113,
or 114 of SEQ ID NO:2; wherein the recombinant TGF-.beta. ligand
polypeptide activates cell proliferation or activity associated
with SMAD pathway.
2. The recombinant polypeptide of claim 1, wherein the third
segment comprises amino acid residues x.sub.17 to x.sub.1 of SEQ ID
NO:2 ("3b"), x.sub.17 is x.sub.16+1 of SEQ ID NO: 2.
3. The recombinant polypeptide of claim 1, wherein the third
segment comprises amino acid residues x.sub.2 to x.sub.3 of SEQ ID
NO:5 ("3a").
4. The recombinant polypeptide of claim 1, wherein the third
segment comprises amino acid residues x.sub.2 to x.sub.3 of SEQ ID
NO:5 ("3a"); the fourth segment comprises amino acid residues
x.sub.4 to x.sub.5 of SEQ ID NO:5 ("4a"), wherein x.sub.6 is
x.sub.3+1 of SEQ ID NO: 5; the fifth segment comprises amino acid
residues x.sub.8 to x.sub.9 of SEQ ID NO:5 ("5a"), wherein x.sub.8
is x.sub.5+1 of SEQ ID NO: 5; and the sixth segment comprises amino
acid residues x.sub.12 to x.sub.13 of SEQ ID NO:5 ("6a"), wherein
x.sub.12 is x.sub.9+1 of SEQ ID NO: 5.
5. The recombinant polypeptide of claim 1, wherein the third
segment comprises amino acid residues x.sub.2 to x.sub.3 of SEQ ID
NO:5 ("3a"); the fourth segment comprises amino acid residues
x.sub.4 to x.sub.5 of SEQ ID NO:5 ("4a"), wherein x.sub.6 is
x.sub.3+1 of SEQ ID NO: 5; the fifth segment comprises amino acid
residues x.sub.10 to x.sub.11 of SEQ ID NO:5 ("5b"), wherein
x.sub.10 is x.sub.7 of SEQ ID NO: 2; and the sixth segment
comprises amino acid residues x.sub.12 to x.sub.13 of SEQ ID NO:5
("6a"), wherein x.sub.12 is x.sub.9 of SEQ ID NO: 5.
6. The recombinant polypeptide of claim 1, wherein the third
segment comprises amino acid residues x.sub.17 to x.sub.1 of SEQ ID
NO:2 ("3b"), x.sub.17 is x.sub.16+1 of SEQ ID NO: 2; the fourth
segment comprises amino acid residues x.sub.6 to x.sub.7 of SEQ ID
NO:2 ("4b"), wherein x.sub.6 is x.sub.1+1 of SEQ ID NO: 2; the
fifth segment comprises amino acid residues x.sub.8 to x.sub.9 of
SEQ ID NO:5 ("5a"), wherein x.sub.8 is x.sub.5 of SEQ ID NO: 5; and
the sixth segment comprises amino acid residues x.sub.12 to
x.sub.13 of SEQ ID NO:5 ("6a"), wherein x.sub.12 is x.sub.9+1 of
SEQ ID NO: 5.
7. The recombinant polypeptide of claim 1, wherein the third
segment comprises amino acid residues x.sub.17 to x.sub.1 of SEQ ID
NO:2 ("3b"), x.sub.17 is x.sub.16+1 of SEQ ID NO: 2; the fourth
segment comprises amino acid residues x.sub.6 to x.sub.7 of SEQ ID
NO:2 ("4b"), wherein x.sub.6 is x.sub.1+1 of SEQ ID NO: 2; the
fifth segment comprises amino acid residues x.sub.10 to x.sub.11 of
SEQ ID NO:2 ("5b"), wherein x.sub.10 is x.sub.7+1 of SEQ ID NO: 2;
and the sixth segment comprises amino acid residues x.sub.12 to
x.sub.13 of SEQ ID NO:5 ("6a"), wherein x.sub.12 is x.sub.9 of SEQ
ID NO: 5.
8. The recombinant polypeptide of claim 1, wherein the third
segment comprises amino acid residues x.sub.17 to x.sub.1 of SEQ ID
NO:2 ("3b"), x.sub.17 is x.sub.16+1 of SEQ ID NO: 2; the fourth
segment comprises amino acid residues x.sub.6 to x.sub.7 of SEQ ID
NO:2 ("4b"), wherein x.sub.6 is x.sub.1+1 of SEQ ID NO: 2; the
fifth segment comprises amino acid residues x.sub.8 to x.sub.9 of
SEQ ID NO:5 ("5a"), wherein x.sub.8 is x.sub.5 of SEQ ID NO: 5; and
the sixth segment comprises amino acid residues x.sub.14 to
x.sub.15 of SEQ ID NO:5 ("6b"), wherein x.sub.14 is x.sub.11 of SEQ
ID NO: 2.
9. The recombinant TGF-.beta. ligand polypeptide of claim 1,
wherein the recombinant TGF-.beta. ligand polypeptide has at least
97% sequence identity to the amino acid sequence comprising the
first, the second, the third, the fourth, the fifth, and the sixth
segments.
10. The recombinant TGF-.beta. ligand polypeptide of claim 1,
wherein the recombinant TGF-.beta. ligand polypeptide has at least
98% sequence identity to the amino acid sequence comprising the
first, the second, the third, the fourth, the fifth, and the sixth
segments.
11. The recombinant TGF-.beta. ligand polypeptide of claim 1,
wherein the recombinant TGF-.beta. ligand polypeptide has at least
99% sequence identity to the amino acid sequence comprising the
first, the second, the third, the fourth, the fifth, and the sixth
segments.
12. The recombinant TGF-.beta. ligand polypeptide of claim 1,
wherein the recombinant TGF-.beta. ligand polypeptide has 100%
sequence identity to the amino acid sequence comprising the first,
the second, the third, the fourth, the fifth, and the sixth
segments.
13. The recombinant TGF-.beta. ligand polypeptide of claim 1,
wherein the polypeptide comprises a sequence selected from the
group consisting of SEQ ID NOs: 13, 15, 9, 7, 11, 114, 116, 118,
120, 122, 124, 126, 128, 130, and 132.
14. The recombinant TGF-.beta. ligand polypeptide of claim 1,
wherein said polypeptide comprises the sequence set forth in SEQ ID
NO: 9.
15. The recombinant TGF-.beta. ligand polypeptide of claim 1,
wherein said polypeptide has at least 95% sequence identity to the
sequence set forth in SEQ ID NO: 9.
16. A polynucleotide comprising a sequence selected from the group
consisting of SEQ ID NOs: 12, 14, 8, 6, 10, 113, 115, 117, 119,
121, 123, 125, 127, 129, and 131.
17. The polynucleotide of claim 11, wherein said polynucleotide has
at least 95% sequence identify to the sequence set forth in SEQ ID
NO: 8.
18. A method of activating cell proliferation or activity
associated with SMAD pathway, comprising contact a cell or tissue
with a recombinant TGF-.beta. ligand polypeptide of claim 1.
19. A method of treating a disease or disorder associated with
bone, cartilage, neurological tissue, cardiac tissue, skeletal
muscle or endocrine tissue in a subject in need of the treatment,
comprising contacting the patient with a recombinant TGF-.beta.
ligand polypeptide of claim 1.
20. A method of treating a cell proliferative disease or disorder
in a subject in need of the treatment, comprising contacting the
patient with a recombinant TGF-.beta. ligand polypeptide of claim
1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The application is a continuation of U.S. patent application
Ser. No. 12/712,069, filed Feb. 24, 2010, which claims priority
under 35 U.S.C. .sctn.119 to U.S. Provisional Application Ser. No.
61/155,066, filed Feb. 24, 2009, the disclosure of the
aforementioned priority applications is incorporated herein by
reference in its entirety.
TECHNICAL FIELD
[0003] The present disclosure relates to biomolecular engineering
and design, and engineered proteins and nucleic acids.
BACKGROUND
[0004] Activins and Bone Morphogenetic Proteins (BMPs) are members
of the much larger Transforming Growth Factor-beta (TGF-.beta.)
superfamily. Due to their pervasiveness in numerous developmental
and cellular processes, TGF-.beta. ligands have been the focus of
great interest. For TGF-.beta. ligands to be successfully used as
therapeutic tools, several hurdles need to be overcome. The ability
to specifically modify and alter the properties of TGF-.beta.
ligands, as well as generate those ligands in significant
quantities is required.
SUMMARY
[0005] The disclosure provides non-naturally occurring chimeric
polypeptides having an activity provided by a TGF-beta family of
proteins. The chimeric polypeptides of the disclosure comprises two
or more segments or fragments from parental TGF-beta proteins
operably linked such that the resulting polypeptide is capable of
modulating a pathway associated with a TGF-beta family of proteins.
In one embodiment, the pathway is a SMAD or DAXX pathway.
[0006] In one embodiment, the disclosure provides designer TGF-beta
ligands that can be synthesized by selecting and conjoining
different sequence segments of TGF-beta superfamily ligands to
construct new ligands (designer ligands). These novel ligands
possess entirely new protein sequence library that differs from
naturally existing TGF-beta superfamily ligands. This approach
originates primarily from the recognition of the structural
commonality among natural TGF-beta superfamily ligands. All
.about.40 TGF-beta superfamily ligands share the same overall
architecture with generic characteristics for each region of the
protein. The framework of TGF-beta ligands can be divided into
(generally) six subdomains (also called sequence segments; marked
in six different colors in FIG. 19) that all superfamily members
share.
[0007] In one embodiment, the disclosure also provides a
recombinant polypeptide comprising: at least two peptide segments,
a first segment of the polypeptide comprising a sequence having at
least 80% identity to a first TGF-beta family protein and a second
segment comprising a sequence having at least 80% identity to a
second TGF-beta family protein, wherein the segments are operably
linked and have activity of at least one of the first or second
parental TGF-beta family protein. In one embodiment, the the
polypeptide comprises an N-terminal segment from BMP-2. In another
embodiment, the at least two peptide segments comprise 6 peptide
segments operably linked N-terminus to C-terminus. In yet another
embodiment, each of the first and second TGF-beta family proteins
have structural similarity and each segment corresponds to a
structural motif. In yet a further embodiment, the first TGF-beta
family protein is BMP-2 and the second TGF-beta family protein is
activin. In one embodiment, the segments of the BMP-2 protein
comprise segment 1: amino acid residue from about 1 to about
x.sub.1 of SEQ ID NO:2 ("1b"); segment 2 is from about amino acid
residue x.sub.1 to about x.sub.2 of SEQ ID NO:2 ("2b"); segment 3
is from about amino acid residue x.sub.2 to about x.sub.3 of SEQ ID
NO:2 ("3b"); segment 4 is from about amino acid residue x.sub.3 to
about x.sub.4 of SEQ ID NO:2 ("4b"); segment 5 is from about amino
acid residue x.sub.4 to about x.sub.5 of SEQ ID NO:2 ("5b"); and
segment 6 is from about amino acid residue x.sub.5 to about x.sub.6
of SEQ ID NO:2 ("6b"); and wherein: x.sub.1 is residue 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, or 35 of SEQ ID NO:2; x.sub.2 is
residue 45, 46, 47, or 48 of SEQ ID NO:2; x.sub.3 is residue 65,
66, 67, or 68 of SEQ ID NO:2; x.sub.4 is residue 76, 77, 78, 79,
80, 81 or 82 of SEQ ID NO:2; x.sub.5 is residue 88, 89, 90, 91, 92,
93, or 94 of SEQ ID NO:2; and x.sub.6 is residue 112, 113, or 114
or SEQ ID NO:2, corresponding to the C-terminus of BMP-2; and the
segments of the activin protein comprise segment 1, amino acid
residue from about 1 to about x.sub.1 of SEQ ID NO:5 ("1a");
segment 2 is from about amino acid residue x.sub.1 to about x.sub.2
of SEQ ID NO:5 ("2a"); segment 3 is from about amino acid residue
x.sub.2 to about x.sub.3 of SEQ ID NO:5 ("3a"); segment 4 is from
about amino acid residue x.sub.3 to about x.sub.4 of SEQ ID NO:5
("4a"); segment 5 is from about amino acid residue x.sub.4 to about
x.sub.5 of SEQ ID NO:5 ("5a"); and segment 6 is from about amino
acid residue x.sub.5 to about x.sub.6 of SEQ ID NO:5 ("6a"); and
wherein: x.sub.1 is residue 22, 23, 24, 25, 26, 27, 28, 29, 30, 31
or 32 of SEQ ID NO:5; x.sub.2 is residue 42, 43, 44, or 45 of SEQ
ID NO:5; x.sub.3 is residue 61, 62, 63, or 64 of SEQ ID NO:5;
x.sub.4 is residue 78, 79, 80, 81, 82, 83 or 84 of SEQ ID NO:5;
x.sub.5 is residue 90, 91, 92, 93, 94, 95 or 96 of SEQ ID NO:5; and
x.sub.6 is residue 114, 115, or 116 or SEQ ID NO:5; and wherein the
polypeptide has an order of segment 1-segment 2-segment 3-segment
4-segment 5-segment 6. In a further embodiment, the polypeptide
comprises a sequence of segments selected from the group consisting
of 1b2b3b4b5b6a; 1b2b3b4b5a6a; 1b2b3b4b5a6b; 1b2b3a4a5a6a;
1b2b3a4a5b6a; 1b2a3a4a5a6a; 1b2a3a4a5a6a L66V/V67I;
1b(1a_II)2a3a4a5a6a; 1b2a3a4a5a6b; 1b2a3a4a5b6b; 1b2a3a4a5b6a;
1b2a3b4b5b6a; 1b2a3b4b5a6a; and 1b2a3b4b5a6b.
[0008] The disclosure also provides a recombinant polypeptide
comprising at least 80%, 90%, 95%, 98% or 99% identity to a
sequence as set forth in SEQ ID NO:7, 9, 11, 13, 15, 17, 19, 1, 23,
25, 2, 29, 31, 33, 35, 37, 39 or 41 and wherein the polypeptide
modulates the SMAD or DAXX pathway.
[0009] The disclosure also provides a chimeric TGF-beta family
polypeptide comprising a segment of a first TGF-beta family protein
operably linked to a segment of a second different TGF-beta family
protein to provide a chimeric polypeptide having SMAD or DAXX
modulating activity.
[0010] The disclosure also provides a polynucleotide encoding a
polypeptide of the disclosure. In one embodiment, the
polynucleotide has at least 80%, 90%, 95%, 98%, 99% or more
identity to a sequence selected from the group consisting of SEQ ID
NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 24, 26,
28, and 40. Vectors comprising such polynucleotides are also
provided along with recombinant cells.
[0011] In an alternate embodiment, the disclosure provides novel
ligands for members of the TGF-beta superfamily, wherein the ligand
is a chimeric protein with at least one of six subdomians from a
foreign or different member of the TGF-beta superfamily.
[0012] The disclosure comprises a chimeric polypeptide comprising a
first domain from a first TGF-beta family member, a crossover point
at J1 (see, e.g., FIG. 18), a second domain from the same or second
TGF-beta family member, a crossover point at J2 (see, e.g., FIG.
18), a third domain from the same or third TGF-beta family member,
a crossover point at J3 (see, e.g., FIG. 18), a fourth domain from
the same or fourth TGF-beta family member, a crossover point at J4
(see e.g., FIG. 18), a fifth domain from the same or fifth TGF-beta
family member, a crossover point at J5 (see, e.g., FIG. 18), and a
second domain from the same or sixth TGF-beta family member. In one
embodiment, the chimera is derived from 2, 3, 4, 5, or 6 different
TGF-beta family members. In yet another embodiment a crossover at
J3 is optional.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A-1B show BMP2/BMP6 sample characterization. FIG. 1A:
BMP2/BMP6 sample on SDS-PAGE. The BMP2/BMP6 sample non-reduced in
lane one, molecular weight marker in lane two, and the BMP2/BMP6
sample reduced in lane three. FIG. 1B: SELDI-TOF-MS overlaid
results from separate samples of BMP2, BMP6, and BMP2/BMP6
ligands.
[0014] FIG. 2 shows C.sub.2Cl.sub.2 whole cell Smad1-dependent
reporter assay with wild type ligands. The solid black bars
represent error.
[0015] FIG. 3 shows a visualization of chick limb bud mesenchyme
cell micromass culture chrondogenesis assays after 5 days. The top
panel shows the culture with no factor and the extracellular
antagonist Noggin. The bars in the micrographs represent 1 mm. The
second through fourth panels show the culture with ligands BMP2,
BMP6, and BMP2/BMP6 respectively.
[0016] FIG. 4 shows quantification of chick limb bud mesenchyme
cell micromass culture chrondogenesis assays after three days. The
addition of the specified growth factor at different concentrations
is indicated.
[0017] FIG. 5 shows Mutant BMP2/BMP6 heterodimers displaying
activation of Smad1 reporter gene. Sample a contains no factor
(background) and Sample b contains a BMP2 homodimer with no active
receptor sites. All quantities are normalized to Sample c which is
the fully active BMP2/BMP6 heterodimer (100% activation of reporter
gene).
[0018] FIG. 6 shows a native-PAGE displaying the quantity of type
II receptor ECD remaining after being saturated into a
ligand-receptor complex with the specified ligands.
[0019] FIGS. 7A-7C depict a structure/sequence aligment for chimera
design strategy. FIG. 7A shows a sequence alignment of BMP-2 and
Activin-.beta.A with the segments highlighted and labeled. FIG. 7B
shows the monomer structure of BMP-2 and FIG. 7C shows the monomer
structure of Activin-.beta.A.
[0020] FIG. 8 provides a graph showing cell viability in the
presence of xerogel material (lowest concentration, green, 0.3
ug/ul; medium concentration, red, 3 ug/ul; high concentration,
blue, 30 ug/ul).
[0021] FIGS. 9A-9F show H9 hES cells cultured in mCIVA using
different concentrations of AB2-008 in the absence or presence of
human FGF2. FIG. 9A shows differentiated H9 cells after 3 passages
(1 ng/mL, AB2-008; no FGF2). FIG. 9B shows differentiated H9 cells
after 3 passages (10 ng/mL, AB2-008; no FGF2). FIG. 9C shows
differentiated H9 cells after 11 passages (100 ng/mL, AB2-008; no
FGF2). FIG. 9D shows differentiated H9 cells after 12 passages (1
ng/mL, AB2-008; 100 ng/mL, FGF2). FIG. 9E shows H9 cells after 13
passages (10 ng/mL, AB2-008; 100 ng/mL, FGF2). And FIG. 9F shows
differentiated H9 cells after 9 passages (100 ng/mL, AB2-008; 100
ng/mL, FGF2). In the figures, differentiated cells are denoted by
arrows.
[0022] FIG. 10 shows mineralization shown by Von Kossa staining
with (A) no ligand added, (B) recombinant BMP2 (30 ng/ml), C)
AB2-004 (30 ng/ml), D) AB2-011 (30 ng/ml) and E) AB2-015 (30
ng/ml).
[0023] FIG. 11 shows the signaling activities of AB2-008, AB2-009,
and AB2-010. (A) AB2-008 versus activin-.beta.A (B) AB2-009 versus
activin-.beta.A (C) AB2-010 versus activin-.beta.A (D) Relative
signaling strength in comparison to activin-.beta.A.
[0024] FIG. 12 shows phosphorylation by AB2-008, AB2-009, and
AB2-010 in comparison to Activin-.beta.A and BMP2. Activin-.beta.A,
AB2-008, and AB2-009 show comparable levels of phosphorylation of
SMAD2, whereas BMP2 shows phosphorylation of SMAD1
specifically.
[0025] FIG. 13 depicts FSH release by Activin-.beta.A, AB2-008,
AB2-009, and AB2-010. (A) Dose dependent FSH stimulation without
Inhibin, and (B) decreased release with Inhibin.
[0026] FIG. 14 shows co-receptor binding by Activin-.beta.A and
AB2-008. Smad-2-Luciferase activity in HEK cells in the presence of
and absence Cripto with (A) activin-.beta.A, and with (B)
AB2-008.
[0027] FIG. 15 shows signaling activity of AB2-011, AB2-012, and
AB2-015 by Smad-1 pathway. (A) AB2-004 v.s. BMP2, (B) AB2-011 v.s.
BMP2, (C) AB2-012 v.s. BMP2, and (D) AB2-015 v.s. BMP2, all in the
concentration range of 3-30 ng/ml in culture media using Smad-1
Luciferase assay with C2C12 cells.
[0028] FIG. 16 shows Noggin sensitivity of BMP2, AB2-004, AB2-011,
AB2-012, and AB2-015. Smad-1 luciferase signaling acitivity is
measured with (light gray) and without (dark gray) Noggin.
[0029] FIG. 17 is a schematic description of the RASCH method.
[0030] FIG. 18 provides an alignment of the sequences of several
members of the TGF-beta superfamily, with the relative segments
defined for each member.
[0031] FIG. 19 shows the six subdomains (fragments) on a single
subunit of the TGF-beta superfamily ligand's scaffold.
DETAILED DESCRIPTION
[0032] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a domain" includes a plurality of such domains and reference to
"the protein" includes reference to one or more proteins, and so
forth.
[0033] Also, the use of "or" means "and/or" unless stated
otherwise. Similarly, "comprise," "comprises," "comprising"
"include," "includes," and "including" are interchangeable and not
intended to be limiting.
[0034] It is to be further understood that where descriptions of
various embodiments use the term "comprising," those skilled in the
art would understand that in some specific instances, an embodiment
can be alternatively described using language "consisting
essentially of" or "consisting of."
[0035] Although methods and materials similar or equivalent to
those described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods and materials are
described herein.
[0036] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs. Thus,
as used throughout the instant application, the following terms
shall have the following meanings.
[0037] "Amino acid" is a molecule having the structure wherein a
central carbon atom (the alpha-carbon atom) is linked to a hydrogen
atom, a carboxylic acid group (the carbon atom of which is referred
to herein as a "carboxyl carbon atom"), an amino group (the
nitrogen atom of which is referred to herein as an "amino nitrogen
atom"), and a side chain group, R. When incorporated into a
peptide, polypeptide, or protein, an amino acid loses one or more
atoms of its amino acid carboxylic groups in the dehydration
reaction that links one amino acid to another. As a result, when
incorporated into a protein, an amino acid is referred to as an
"amino acid residue."
[0038] "Protein" or "polypeptide" refers to any polymer of two or
more individual amino acids (whether or not naturally occurring)
linked via a peptide bond, and occurs when the carboxyl carbon atom
of the carboxylic acid group bonded to the alpha-carbon of one
amino acid (or amino acid residue) becomes covalently bound to the
amino nitrogen atom of amino group bonded to the -carbon of an
adjacent amino acid. The term "protein" is understood to include
the terms "polypeptide" and "peptide" (which, at times may be used
interchangeably herein) within its meaning. In addition, proteins
comprising multiple polypeptide subunits (e.g., DNA polymerase III,
RNA polymerase II) or other components (for example, an RNA
molecule, as occurs in telomerase) will also be understood to be
included within the meaning of "protein" as used herein. Similarly,
fragments of proteins and polypeptides are also within the scope of
the disclosure and may be referred to herein as "proteins." In one
aspect of the disclosure, a polypeptide comprises a chimera of two
or more parental peptide segments.
[0039] As used herein, TGF-beta superfamily member refers to a
TGF-beta superfamily (including bone morphogenic factors) gene or
protein of any species, particularly a mammalian species, including
but not limited to bovine, ovine, porcine, murine, equine, and
human. "TGF-beta superfamily polypeptide" refers to the amino acid
sequences of purified TGF-beta superfamily protein obtained from
any species, particularly a mammalian species, including bovine,
ovine, porcine, murine, equine, and human and from any source,
whether natural, synthetic, semi-synthetic, or recombinant.
[0040] "Peptide segment" refers to a portion or fragment of a
larger polypeptide or protein. A peptide segment need not on its
own have functional activity, although in some instances, a peptide
segment may correspond to a domain of a polypeptide wherein the
domain has its own biological activity. A stability-associated
peptide segment is a peptide segment found in a polypeptide that
promotes stability, function, or folding compared to a related
polypeptide lacking the peptide segment.
[0041] A particular amino acid sequence of a given protein (i.e.,
the polypeptide's "primary structure," when written from the
amino-terminus to carboxy-terminus) is determined by the nucleotide
sequence of the coding portion of a mRNA, which is in turn
specified by genetic information, typically genomic DNA (including
organelle DNA, e.g., mitochondrial or chloroplast DNA). Thus,
determining the sequence of a gene assists in predicting the
primary sequence of a corresponding polypeptide and more particular
the role or activity of the polypeptide or proteins encoded by that
gene or polynucleotide sequence.
[0042] "Fused," "operably linked," and "operably associated" are
used interchangeably herein to broadly refer to a chemical or
physical coupling of two otherwise distinct domains, wherein each
domain has independent biological function. As such, the present
disclosure provides TGF-beta (e.g., BMP or activins) domains that
are fused to one another such that they function as a polypeptide
having a TGF-beta family activity or an improvement or change in
ligand specificity of a TGF-beta family of polypeptides. In one
embodiment, a chimeric polypeptide comprising a plurality of
domains from two parental TGF-beta family polypeptides are linked
such that they are part of the same coding sequence, each domain
encoded by a polynucleotide from a parental TGF-beta family
polypeptide, wherein the polynucleotides are in frame such that the
polynucleotide when transcribed encodes a single mRNA that when
translated comprises a plurality of domains as a single
polypeptide. Typically, the coding domains will be linked
"in-frame" either directly of separated by a peptide linker and
encoded by a single polynucleotide. Various coding sequences for
peptide linkers and peptide are known in the art.
[0043] "Polynucleotide" or "nucleic acid" refers to a polymeric
form of nucleotides. In some instances a polynucleotide comprises a
sequence that is not immediately contiguous with either of the
coding sequences with which it is immediately contiguous (one on
the 5' end and one on the 3' end) in the naturally occurring genome
of the organism from which it is derived. The term therefore
includes, for example, a recombinant DNA which is incorporated into
a vector; into an autonomously replicating plasmid or virus; or
into the genomic DNA of a prokaryote or eukaryote, or which exists
as a separate molecule (e.g., a cDNA) independent of other
sequences. The nucleotides of the disclosure can be
ribonucleotides, deoxyribonucleotides, or modified forms of either
nucleotide. A polynucleotides as used herein refers to, among
others, single- and double-stranded DNA, DNA that is a mixture of
single- and double-stranded regions, single- and double-stranded
RNA, and RNA that is mixture of single- and double-stranded
regions, hybrid molecules comprising DNA and RNA that may be
single-stranded or, more typically, double-stranded or a mixture of
single- and double-stranded regions. The term polynucleotide
encompasses genomic DNA or RNA (depending upon the organism, i.e.,
RNA genome of viruses), as well as mRNA encoded by the genomic DNA,
and cDNA.
[0044] "Nucleic acid segment," "oligonucleotide segment" or
"polynucleotide segment" refers to a portion of a larger
polynucleotide molecule. The polynucleotide segment need not
correspond to an encoded functional domain of a protein; however,
in some instances the segment will encode a functional domain of a
protein. A polynucleotide segment can be about 6 nucleotides or
more in length (e.g., 6-20, 20-50, 50-100, 100-200, 200-300,
300-400 or more nucleotides in length).
[0045] "Chimera" or "chimeric protein" or "chimeric polypeptide"
refers to a combination of at least two segments of at least two
different parent proteins. As appreciated by one of skill in the
art, the segments need not actually come from each of the parents,
as it is the particular sequence that is relevant, and not the
physical nucleic acids themselves. For example, a chimeric BMP will
have at least two segments from two different parent BMPs; or BMP
and other member of the TGF-beta superfamily, or alternatively, an
unrelated protein. A chimeric protein may also be an
"interspecies," "intergenic," etc. fusion of protein structures
(the same or different member protein) expressed by different kinds
of organisms. In a one embodiment, two segments are connected so as
to result in a new chimeric protein. In other words, a protein will
not be a chimera if it has the identical sequence of either one of
the full-length parents. A chimeric protein can comprise more than
two segments from two different parent proteins. For example, there
may be 2, 3, 4, 5, 6 or 10-20, or more parents from which the
domains may be derived in generating a final chimera or library of
chimeras. The segment of each parent protein can be very short or
very long, the segments can range in length of contiguous amino
acids from 1 to about the full length of the protein. In one
embodiment, the minimum length is 5 amino acids. Generally, the
segment or subdomain, is one of six subdomains, alternatively five
subdomains (see FIGS. 18 and 19). The six segments of a TGF-beta
superfamily member are identified based on the structural
architecture of the member protein and/or the primary amino acid
sequence as aligned against other homologous member proteins. As
identified, the member protein is generally divided into 6 distinct
sections (although, alternatively, 5 distinct sections) based on
segments derived to minimize alterations, or alternatively viewed,
maximize alterations, to the aligned native TGF-beta member
sequence during chimera engineering. Generally, FIG. 18 shows the
relative positions of the distinct segments overlapping the aligned
sequences of each of several TGF-beta superfamily members. The
vertical line denotes a general position for cross-over between
domains in generating the chimera. The amino acids that can overlap
the two domains can be defined as being plus or minus about 5 amino
acids (or alternatively, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids) in
either direction of the vertical line. Also in FIG. 18 is shown a
boxed set of amino acids that identify additionl junctions that can
be used to generate chimera. The J1-J5 junctions are positions
general conservation across the TGF-beta family proteins that can
be used to generate cross-over points.
[0046] Although relatively distinct, the segments may comprise a
particular amino acid sequence or an original amino acid sequence
that is amenable to substitution(s), insertion(s), additional amino
acid(s) at either or both termini of the original sequence, or
other modifications. By "amenable", it is meant that the structural
integrity of each segment is maintained as compared to the domain
of the original sequence. For example, a segment described herein
of a TGF-beta superfamily member may shift by 10, 5, 3, 2, or 1, or
preferably no more than 1 amino acid on either or both termini of
segment as identified.
[0047] In one embodiment, the disclosure provides a chimeric
protein comprising a fusion of at least one segment from a TGF-beta
member with a second segment from a second TGF-beta member, wherein
the first segment is foreign to the second TGF-beta member.
Utilizing the five-six subdomains (segments) on a single subunit of
the TGF-beta superfamily ligand as a scaffold framework, new
(designer) sequences can be recombinantly linked by mixing segments
from different TGF-beta ligands in the same order as they appear in
nature. This assembly produces new sequences that are partly
similar to one of several different target sequences, but
distinctly different from any naturally occurring sequences.
[0048] In one embodiment, a single crossover point is defined for
two parents. The crossover location defines where one parent's
amino acid segment will stop and where the next parent's amino acid
segment will start. Thus, a simple chimera would only have one
crossover location where the segment before that crossover location
would belong to one parent and the segment after that crossover
location would belong to the second parent. In one embodiment, the
chimera has more than one crossover location. For example, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11-30, or more crossover locations. In a
particular embodiment, the parental strands are defined as having 4
or 5 crossover locations. How these crossover locations are named
and defined are both discussed below. In an embodiment where there
are two crossover locations and two parents, there will be a first
contiguous segment from a first parent, followed by a second
contiguous segment from a second parent, followed by a third
contiguous segment from the first parent. Contiguous is meant to
denote that there is nothing of significance interrupting the
segments. These contiguous segments are connected to form a
contiguous amino acid sequence. For example, a BMP-2/activin
chimera derived from a BMP-2 wild-type parental strand and an
activin wild-type parental strand with two crossovers would
comprise a first segment from either BMP-2 or activin, a second
segment from the opposite parental strand compared to the first
segment operably linked to the first strand and comprising a
structural motif downstream of the first strand and a third segment
strand from the opposite parental strand compared to the second
segment and from the same parental strand as the first segment
operably linked to the second strand and comprising a structural
motif downstream of the second strand all connected in one
contiguous amino acid chain.
[0049] As appreciated by one of skill in the art, variants of
chimeras exist as well as the exact sequences. In otherwords
conservative amino acid substitutions may be incorporated into the
chimera (e.g., from about 1-10 conservative amino acid
substitutions). Thus, not 100% of each segment need be present in
the final chimera if it is a variant chimera. The amount that may
be altered, either through additional residues or removal or
alteration of residues will be defined as the term variant is
defined. Of course, as understood by one of skill in the art, the
above discussion applies not only to amino acids but also nucleic
acids which encode for the amino acids.
[0050] "Conservative amino acid substitution" refers to the
interchangeability of residues having similar side chains, and thus
typically involves substitution of the amino acid in the
polypeptide with amino acids within the same or similar defined
class of amino acids. By way of example and not limitation, an
amino acid with an aliphatic side chain may be substituted with
another aliphatic amino acid, e.g., alanine, valine, leucine,
isoleucine, and methionine; an amino acid with hydroxyl side chain
is substituted with another amino acid with a hydroxyl side chain,
e.g., serine and threonine; an amino acids having aromatic side
chains is substituted with another amino acid having an aromatic
side chain, e.g., phenylalanine, tyrosine, tryptophan, and
histidine; an amino acid with a basic side chain is substituted
with another amino acid with a basis side chain, e.g., lysine,
arginine, and histidine; an amino acid with an acidic side chain is
substituted with another amino acid with an acidic side chain,
e.g., aspartic acid or glutamic acid; and a hydrophobic or
hydrophilic amino acid is replaced with another hydrophobic or
hydrophilic amino acid, respectively.
[0051] "Non-conservative substitution" refers to substitution of an
amino acid in the polypeptide with an amino acid with significantly
differing side chain properties. Non-conservative substitutions may
use amino acids between, rather than within, the defined groups and
affects (a) the structure of the peptide backbone in the area of
the substitution (e.g., proline for glycine) (b) the charge or
hydrophobicity, or (c) the bulk of the side chain. Byway of example
and not limitation, an exemplary non-conservative substitution can
be an acidic amino acid substituted with a basic or aliphatic amino
acid; an aromatic amino acid substituted with a small amino acid;
and a hydrophilic amino acid substituted with a hydrophobic amino
acid.
[0052] "Reference sequence" refers to a defined sequence used as a
basis for a sequence comparison. A reference sequence may be a
subset of a larger sequence, for example, a segment of a
full-length gene or polypeptide sequence. Generally, a reference
sequence can be at least 20 nucleotide or amino acid residues in
length, at least 25 residues in length, at least 50 residues in
length, or the full length of the nucleic acid or polypeptide.
Since two polynucleotides or polypeptides may each (1) comprise a
sequence (i.e., a portion of the complete sequence) that is similar
between the two sequences, and (2) may further comprise a sequence
that is divergent between the two sequences, sequence comparisons
between two (or more) polynucleotides or polypeptides are typically
performed by comparing sequences of the two polynucleotides or
polypeptides over a "comparison window" to identify and compare
local regions of sequence similarity.
[0053] "Sequence identity" means that two amino acid sequences are
substantially identical (i.e., on an amino acid-by-amino acid
basis) over a window of comparison. The term "sequence similarity"
refers to similar amino acids that share the same biophysical
characteristics. The term "percentage of sequence identity" or
"percentage of sequence similarity" is calculated by comparing two
optimally aligned sequences over the window of comparison,
determining the number of positions at which the identical residues
(or similar residues) occur in both polypeptide 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 (i.e., the window size), and multiplying the result by
100 to yield the percentage of sequence identity (or percentage of
sequence similarity). With regard to polynucleotide sequences, the
terms sequence identity and sequence similarity have comparable
meaning as described for protein sequences, with the term
"percentage of sequence identity" indicating that two
polynucleotide sequences are identical (on a
nucleotide-by-nucleotide basis) over a window of comparison. As
such, a percentage of polynucleotide sequence identity (or
percentage of polynucleotide sequence similarity, e.g., for silent
substitutions or other substitutions, based upon the analysis
algorithm) also can be calculated. Maximum correspondence can be
determined by using one of the sequence algorithms described herein
(or other algorithms available to those of ordinary skill in the
art) or by visual inspection.
[0054] As applied to polypeptides, the term substantial identity or
substantial similarity means that two peptide sequences, when
optimally aligned, such as by the programs BLAST, GAP or BESTFIT
using default gap weights or by visual inspection, share sequence
identity or sequence similarity. Similarly, as applied in the
context of two nucleic acids, the term substantial identity or
substantial similarity means that the two nucleic acid sequences,
when optimally aligned, such as by the programs BLAST, GAP or
BESTFIT using default gap weights (described in detail below) or by
visual inspection, share sequence identity or sequence
similarity.
[0055] One example of an algorithm that is suitable for determining
percent sequence identity or sequence similarity is the FASTA
algorithm, which is described in Pearson, W. R. & Lipman, D.
J., (1988) Proc. Natl. Acad. Sci. USA 85:2444. See also, W. R.
Pearson, (1996) Methods Enzymology 266:227-258. Preferred
parameters used in a FASTA alignment of DNA sequences to calculate
percent identity or percent similarity are optimized, BL50 Matrix
15: -5, k-tuple=2; joining penalty=40, optimization=28; gap penalty
-12, gap length penalty=-2; and width=16.
[0056] Another example of a useful algorithm is PILEUP. PILEUP
creates a multiple sequence alignment from a group of related
sequences using progressive, pairwise alignments to show
relationship and percent sequence identity or percent sequence
similarity. It also plots a tree or dendogram showing the
clustering relationships used to create the alignment. PILEUP uses
a simplification of the progressive alignment method of Feng &
Doolittle, (1987) J. Mol. Evol. 35:351-360. The method used is
similar to the method described by Higgins & Sharp, CABIOS
5:151-153, 1989. The program can align up to 300 sequences, each of
a maximum length of 5,000 nucleotides or amino acids. The multiple
alignment procedure begins with the pairwise alignment of the two
most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. Using PILEUP, a reference sequence is
compared to other test sequences to determine the percent sequence
identity (or percent sequence similarity) relationship using the
following parameters: default gap weight (3.00), default gap length
weight (0.10), and weighted end gaps. PILEUP can be obtained from
the GCG sequence analysis software package, e.g., version 7.0
(Devereaux et al., (1984) Nuc. Acids Res. 12:387-395).
[0057] Another example of an algorithm that is suitable for
multiple DNA and amino acid sequence alignments is the CLUSTALW
program (Thompson, J. D. et al., (1994) Nuc. Acids Res.
22:4673-4680). CLUSTALW performs multiple pairwise comparisons
between groups of sequences and assembles them into a multiple
alignment based on sequence identity. Gap open and Gap extension
penalties were 10 and 0.05 respectively. For amino acid alignments,
the BLOSUM algorithm can be used as a protein weight matrix
(Henikoff and Henikoff, (1992) Proc. Natl. Acad. Sci. USA
89:10915-10919).
[0058] FIG. 18, for example, shows an alignment of a number of
TGF-beta family members. One of skill in the art can readily
determine from the alignment those amino acids that are conserved
across the family as well as those that are not conserved.
[0059] "Functional" refers to a polypeptide which possesses either
the native biological activity of the naturally-produced proteins
of its type, or any specific desired activity, for example as
judged by its ability to bind to ligand or cognate molecules or
induce a particular biological function (e.g., stimulate muscle
growth, bone growth and the like).
[0060] The Transforming Growth Factor-beta (TGF-.beta.) superfamily
of proteins is comprised of extracellular cytokines found in the
vast majority of human cells. The TGF-.beta. superfamily ligands,
of which there are .about.40, can be subdivided into smaller
families including TGF-.beta., Bone Morphogenetic Proteins (BMPs),
activin and inhibin, Growth and Differentiation Factors (GDFs),
Nodal, Mullerian Inhibiting Substance (MIS), and Glial cell
line-Derived Neurotrophic Factors (GDNFs). TGF-.beta. superfamily
members are found in a diverse range of cell types and play roles
in many fundamental cellular events including dorsal/ventral
patterning and left/right axis determination to bone formation and
tissue repair. More recently, several TGF-.beta. ligands have been
shown to be involved in the maintenance or direct the
differentiation of stem cells. Due to their pervasiveness,
regulation of TGF-.beta. ligand signaling holds promise for the
treatment of a wide range of different diseases from skeletal and
muscle abnormalities to numerous neoplastic events. Exemplary
sequences are provided herein for various members of this family or
proteins, however, one of skill in the art can easily identify
homologs and variants using publicly available databases byword
search or sequence BLAST searches.
[0061] There are generally recognized several subfamilies within
the superfamily of TGF-beta (TGF-.beta.1-.beta.5) as well as the
differentiation factors (e.g., Vg-1), the hormones activin and
inhibin, the Mullerianinhibiting substance (MIS), osteogenic and
morphogenic proteins (e.g., OP-1, OP-2, OP-3, other BMPs), the
developmentally regulated protein Vgr-1, the growth/differentiation
factors (e.g., GDF-1, GDF-3, GDF-9 and dorsalin-1), etc. See, e.g.,
Spom and Roberts (1990) in Peptide Growth Factors and Their
Receptors, Sporn and Roberts, eds., Springer-Verlag: Berlin pp.
419-472; Weeks and Melton (1987) Cell 51: 861-867; Padgett et al.
(1987) Nature 325: 81-84; Mason et al. (1985) Nature 318: 659-663;
Mason et al. (1987) Growth Factors 1: 77-88; Cate et al. (1986)
Cell 45: 685-698; PCT/US90/05903; PCT/US91/07654; PCT/WO94/10202;
U.S. Pat. Nos. 4,877,864; 5,141,905; 5,013,649; 5,116,738;
5,108,922; 5,106,748; and 5,155,058; Lyons et al. (1989) Proc.
Natl. Acad. Sci. USA 86: 4554-58; McPherron et al. (1993) J. Biol.
Chem. 268: 3444-3449; Basler et al. (1993) Cell 73: 687-702.
[0062] Morphogenic proteins of the TGF-beta superfamily include the
mammalian osteogenic protein-1 (OP-1, also known as BMP-7),
osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic
protein-3 (OP3), BMP-2 (also known as BMP-2A or CBMP-2A, and the
Drosophila homolog DPP), BMP-3, BMP-4 (also known as BMP-2B or
CBMP-2B), BMP-5, BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10,
BMP-11, BMP-12, GDF3 (also known as Vgr2), GDF-8, GDF-9, GDF-10,
GDF-11, GDF-12, BMP-13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1
or MP52), GDF-6 (also known as CDMP-2 or BMP13), GDF-7 (also known
as CDMP-3 or BMP-12), the Xenopus homolog Vgl and NODAL, UNIVIN,
SCREW, ADMP, NEURAL, etc.
[0063] One major roadblock for research involving many of the
TGF-.beta. superfamily ligands has been the inability to generate
significant quantities of the proteins. While, BMP-2 is known to
refold efficiently in vitro, other TGF-.beta. ligands (activin,
Nodal, and BMP-7 for instance) have not shown the same refolding
properties. Other expression systems are available to obtain
functional TGF-.beta. ligands. Activin, for example, is expressed
using stably transfected cell lines, such as CHO or transiently
transfected cell lines, such as HEK293 cells.
[0064] The largest sub-family of the TGF-.beta. superfamily is the
BMP/GDF family, which comprises nearly half of all known ligands.
Many of the ligands in BMP/GDF family share both BMP and GDF
designations, such as GDF-7 which also referred to as BMP-12. In
conjunction with being largest family, the BMP/GDF family is also
the most extensively studied family. For example, x-ray crystal
structures have been solved for BMP-2 alone, bound to its type I
receptor, or as a ternary complex bound to both its receptor types.
Additionally, BMP-2, along with BMP-7, has been utilized as an
effective treatment for certain bone injuries. One of the reasons
for the large amount of structural and therapeutic work involving
the BMP/GDF family has been the ability to chemically refold these
ligands. Indeed, BMP-2 is one of the most successful TGF-.beta.
ligands at refolding, with optimized conditions reported to yield
up to 63% active dimer from starting material. However, if specific
amino acids of BMP-2 could be incorporated into other TGF-.beta.
ligands, it may allow for these otherwise non-refoldable ligands to
be refolded opening up the remainder of the TGF-.beta. superfamily
to be better studied.
[0065] TGF-.beta. ligands are synthesized as inactive precursor
molecules composed of an N-terminal pro-domain and a C-terminal
mature domain linked by a protease cleavage site. To be become
active, the mature domain must be cleaved from the pro-domain,
commonly by a convertase, such as furin. Members of the TGF-.beta.
superfamily are classified together due to the conserved structural
architecture found in their mature domains. In general, each mature
ligand monomer contains 7 cysteines, 6 of which form three
intra-disulfide bonds arranged in a `cystine knot` motif.
Stretching outward from the `cystine knot` are 4 beta strands,
creating 2 curved fingers. The last remaining cysteine forms an
inter-disulfide bond with a second ligand monomer, generating a
covalently linked dimer. The dimer has the overall appearance of a
butterfly with the `cystine knot` as the body and the fingers
spreading out like wings. The functional subunit for the TGF-.beta.
superfamily is the dimer and they been shown to exist both as homo-
and heterodimers in vivo. Some family members, such as GDF-9 and
BMP-15, lack the cysteine required to form the inter-disulfide bond
yet they are still able to form stable dimers.
[0066] To initiate the signaling process, TGF-.beta. dimers must
recruit two sets of receptors, termed type I and type II. These
receptors are serine/threonine kinases possessing an extracellular
domain (ECD) ordered into a three-finger toxin fold, a single
transmembrane domain, and a large intracellular kinase domain.
TGF-.beta. ligands have been shown to display preferences in their
affinity for the different receptor types. Activin and Nodal
exhibit high affinity for type II receptors, while BMP-2 and GDF-5
possess higher affinity for type I receptors. Following the binding
of two high affinity receptors to a TGF-.beta. ligand, two lower
affinity receptors are then able to bind and join the complex. Upon
binding of all four receptors to the TGF-.beta. ligand, forming a
6-member ternary complex, the downstream signaling cascade is
initiated. The constitutively active type II receptors
phosphorylate the type I receptors which, in turn, bind and
phosphorylate intracellular signaling molecules called Smads. The
Smad molecules then are able to translocate to the nucleus and
interact directly with transcriptional regulators. Multiple
mechanisms are employed to closely regulate TGF-.beta. signaling at
different stages of the cascade: Extracellular antagonists,
including Noggin, follistatin, and Inhibin; pseudo-receptors
lacking the intracellular kinase domain, similar to BAMBI; or
through intracellular molecules, such as inhibitory Smads.
[0067] TGF-.beta. superfamily shows a high degree of promiscuity by
receptors for the ligands. While there are over 40 TGF-.beta.
ligands, there are only 12 receptors (7 type I and 5 type II).
Therefore, receptors must be able to interact with a multitude of
different ligands. For instance, ActRII is known to bind activin
and BMP-7 with high affinity, but binds BMP-2 with much lower
affinity. In GDF-5, a single amino acid has been found which
determines its type I receptor preference, while in BMP-3 a single
point mutation was discovered which alters type II receptor
affinity as well as imparting function to the ligand. The
disclosure provides methods to create modified TGF-.beta. ligands
with novel receptor binding properties thereby diversifying
TGF-.beta. ligand function as well as compositions having such
activity.
[0068] The disclosure demonstrates a TGF-beta signaling complex by
utilizing novel ligand constructs. Using synthesized chimeric homo-
or heterodimeric ligands the ligands the disclosure provides
compositions for use in dissecting the signaling of TGF-beta family
proteins. Furthermore, utilizing such ligands allows a method for
distinguishing contributions of two type I receptor interfaces from
each other, and two type II receptor interfaced each other. The
methods and compositions of the disclosure demonstrate a
correlation between ligand-receptor affinity, signaling activity,
and biological activity. The methods and compositions of the
disclosure shed light on the mechanism and requirements of the
TGF-beta superfamily signaling complex assembly. In addition the
chimeric ligands provide novel polypeptide for use in treating
diseases and disorders associated with TGF-.beta. family of
proteins.
[0069] The disclosure provides methods of making and novel chimeric
TGF-.beta. ligands which possess the ability to be expressed and
refolded using, for example, an E. coli or mammalian expression
system. These chimeras either mimic a specific TGF-.beta. ligand's
signaling characteristics or display unique signaling properties
not seen in nature. In one embodiment, the disclosure uses
activin-.beta.A and BMP-2 as a template to generate an
activin/BMP-2 chimera with the refolding efficiency of BMP-2 and
the signaling properties of activin-.beta.A; however it should be
recognized that any number of TGF-beta protein family members can
be used. The chimera design scheme of the disclosure yielded
additional TGF-beta member chimera (e.g., activin/BMP-2) ligands
with unnatural signaling characteristics and biological activity.
Such chimeric TGF-beta family polypeptides expand the library of
TGF-.beta. ligands available for structural studies as well as
facilitate the development of novel TGF-.beta. ligands as
therapeutic agents.
[0070] In one embodiment the disclosure provides a series of
activin/BMP-2 chimeric ligands which possess unique signaling
properties. For example, an activin/BMP-2 ligand of the disclosure
exhibited the refolding characteristics of wild type BMP-2 while
retaining activin-like signaling attributes in both in vitro and in
vivo studies. Further, `super` ligands were generated which are
more potent than wild type BMP-2 and were not inhibited by the BMP
antagonist Noggin. The disclosure also provides chimeric TGF-beta
polypeptides comprising an N-terminus of wild type BMP-2mq operably
linked to a different TGF-.beta. ligand polypeptide segment. The
disclosure demonstrates that the N-terminal portion of wild type
BMP-2mq is enough to switch a previously non-refolding ligand into
a refoldable ligand. These findings highlight a method for
obtaining activin and the other TGF-.beta. ligand mimics and
indicate how this strategy can be utilized to expand the library of
TGF-.beta. ligands by diversifying their functionality and promote
the development of unnatural ligands for therapeutic purposes.
[0071] The nucleic acid sequences and polypeptide sequences of
BMP-2 and naturally occurring variants are known. A wild-type BMP-2
nucleic acid sequence (SEQ ID NO:1) and polypeptide (SEQ ID NO:2)
from Rattus sp. are provided. Met at the position N-terminal to the
residue 1 (Q) results from translation of the bacterial initiation
codon (ATG). Furthermore activins are also known in the art (see,
e.g., SEQ ID NO:5). The disclosure provides a number of chimeric
TGF-beta family polypeptides having at least one N-terminal domain
from a BMP-2 and at lease one second domain form another TGF-beta
family members wherein the chimeric polypeptide display activities
different than wild-type parental proteins.
[0072] In one embodiment, two factors were considered when looking
to design the segments of the chimeras. First was a structural
consideration. The overall TGF-.beta. monomer fold is divided into
6 sections naturally: Beta strand 1 and 2, the pre-helix loop,
alpha helix 1, and beta strand 3 and 4. The identification and
characterization of these subdomains are further described in
Example 4. The disclosure utilized a chimeric structures to mimic
these natural regions in the design. Thus, each segment can be
indicated by 1, 2, 3, 4, 5, and 6. The second consideration was to
minimize alterations to the aligned native TGF-beta member sequence
during chimera engineering. Therefore, those regions with sequence
identity between the 2 protein sequences were identified as
putative cross-over points. These regions are suitable for the
overlaps in DNA sequence for PCR strategy and will minimize any
changes to the natural sequences. FIG. 7 illustrates the sequence
and structure of these considerations. The regions are boxed and
numbered according to their section and are mapped onto the ligand
monomer. The areas which can be used for the cross-over points as
segmental boundaries are shaded as a sequence range in orange.
Residue numbering in one embodiment is based on BMP-2 (SEQ ID
NO:2). Thus, cross-over points in generating a chimeric polypeptide
of the disclosure can be identified by identifying similar
struactural motifs in combination with at least 60%, 70%, 80%, 90%,
95%, 98%, 99% or 100% identity in a segment of the sequence between
changes in the structural motif. Cross-overs at these regions
(which may be between 3 to 20 amino acids) minimize disruption of
the resulting chimeric polypeptide providing a stabilized
chimera.
[0073] For example, a chimeric polypeptide comprising the algorithm
1b2b3b4b5b6a indicates 6 segments, the letter indicating the
parental strand of each segment. Thus, in the example
"1b2b3b4b5b6a", segment 1 is from parental strand "b" for BMP-2mq,
segment 2 is from parental strand "b" for BMP-2mq, segment 3 is
from parental strand "b" for BMP-2mq, segment 4 is from parental
strand "b" for BMP-2mq, segment 5 is from parental strand "b" for
BMP-2mq, and segment 6 is from parental strand "a" for Activin.
[0074] In one embodiment, crossover between segments of BMP-2mq and
a second TGF-beta family protein can occur where structural
similarity and sequence similary overlap. FIG. 7 depicts such an
overlap between BMP-2mq and activin, wherein crossovers can be
generated between about residue D25-P35, G45-P48; T65-N68; K76-T82;
and S88-E94 (residue numbering is based on BMP-2 (SEQ ID NO:2)).
Sequence alignment of BMP-2 and activin-.beta.A highlight the
boundaries of segments 1 through 6. Activin has the extra disulfide
bond formed between two Cys. Red (or first shaded boxes on lower
sequences) box notes two amino acids of AB2-009 swapped into
AB2-008. Blue (second shaded box L/Y on lower lines) box notes one
amino acid changed in Segment 5 of BMP2 for all chimera. For
clarity, BMP-2's Segment 5 contains YYD instead of YLD. Green
(KKQ-FFVSFKDI) box denotes a segment introduced into AB2-008 to
make AB2-010, marked as (1a_II) of AB2-010.
[0075] FIG. 18 further depicts such crossover regions with
reference to additional members of the TGF-.beta. family or
proteins. For example, with reference to FIG. 18, one of skill in
the art can see that BMP-3 (SEQ ID NO:43) comprises 110 amino
acids. The first vertical line demonstrates a general region of
cross over and can comprise from 1-5 amino acids on either side of
the vertical line. Accordingly, a first domain from BMP-3 can
comprise amin acid 1 to about x.sub.1, wherein x.sub.1 is an amino
acid corresponding to residue 20-29 (e.g., x1 is 20, 21, 22, 23,
24, 25, 26, 27, 28, or 29). As further shown in FIG. 18, "J1"
corresponds to residues 20-23 of SEQ ID NO:43). J1 refers to a
junction region having conservation across the various species in
the TGF-.beta. family of proteins. Accordingy, a first domain of
BMP-3 comprises amino acids 1 to about x.sub.1, wherein x.sub.1
will be either V or G and the following chimeric domain from a
second TGF-.beta. family member will begin with either G or W.
Using FIG. 18 as a template one of skill in the art can readily
identify the cross-over regions (or junctions points) for the
various members of the TGF-.beta. family. It is important to note
that not every chimera is required to have 6 distinct domains. For
example, a cross over at junction 3 (J3) may not be necessary such
that only 5 or fewer domains from distinct family member are
present in the final chimera.
[0076] Other methods for identifying crossover locations may be
employed in the generation of chimeric TGF-beta family
polypeptides. For example, SCHEMA is a computational based method
for predicting which fragments of homologous proteins can be
recombined without affecting the structural integrity of the
protein (see, e.g., Meyer et al., (2003) Protein Sci.,
12:1686-1693). Chimeras with higher stability are identifiable by
determining the additive contribution of each segment to the
overall stability, either by use of linear regression of
sequence-stability data, or by reliance on consensus analysis of
the MSAs of folded versus unfolded proteins. SCHEMA recombination
ensures that the chimeras retain biological function and exhibit
high sequence diversity by conserving important functional residues
while exchanging tolerant ones.
[0077] As presented in this disclosure, it has been found that when
these recombined, functional chimeric TGF-beta family polypeptides
are generated their ligand specificity can be improved or
biological activity can be altered or improved compared to a
unrecombined parental polypeptide. Because of differences in
activity/ligand profiles, these engineered chimeric TGF-beta family
polypeptides provide a unique basis to screen for activities for
ligand specific activation and inhibition, provide novel
therapeutic polypeptides and research reagents.
[0078] For example, in the chimeras of the disclosure, domain 1, 2,
3, 4, 5, and 6 can be selected from the following sequences (Table
A) wherein the polypeptide comprises a structure (domain 1-domain
2-domain 3-domain 4-domain 5-domain 6):
TABLE-US-00001 TABLE A Amino acids (domain #) SEQ ID NO: Variable
definition 1-x.sub.1 2 x.sub.1 is 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, or 35 (1) 1-x.sub.1 5 x.sub.1 is 22, 23, 24, 25, 26, 27,
28, 29, 30, 31 or 32 (1) 1-x.sub.1 43 x.sub.1 is 20, 21, 22, 23,
24, 25, 26, 27, 28, or 29 (1) 1-x.sub.1 45 x.sub.1 is 27, 28, 29,
30, 31, 32, 33, 34, 35, or 36 (1) 1-x.sub.1 47 x.sub.1 is 43, 44,
44, 46 47, 48, 49, 50, 51, or 52 (1) 1-x.sub.1 49 x.sub.1 is 43,
44, 44, 46 47, 48, 49, 50, 51, or 52 (1) 1-x.sub.1 51 x.sub.1 is
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 (1) 1-x.sub.1 53
x.sub.1 is 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 (1)
1-x.sub.1 55 x.sub.1 is 19, 20, 21, 22, 23, 24, 25, 26 27, or 28
(1) 1-x.sub.1 57 x.sub.1 is 18, 19, 20, 21, 22, 23, 24, 25, 26 or
27 (1) 1-x.sub.1 59 x.sub.1 is 36, 37, 38, 29, 40, 41, 42, 43, 44,
or 45 (1) 1-x.sub.1 61 x.sub.1 is 25, 26, 27, 28, 29, 30, 31, 32,
33 or 34 (1) 1-x.sub.1 63 x.sub.1 is 25, 26, 27, 28, 29, 30, 31,
32, 33 or 34 (1) 1-x.sub.1 65 x.sub.1 is 30, 31, 32, 33, 34, 35,
36, 37, 38, 39 or 40 (1) 1-x.sub.1 67 x.sub.1 is 30, 31, 32, 33,
34, 35, 36, 37, 38, 39 or 40 (1) 1-x.sub.1 69 x.sub.1 is 40, 41,
42, 43, 44, 45, 46, 47, 48 or 49 (1) 1-x.sub.1 71 x.sub.1 is 25,
26, 27, 28, 29, 30, 31, 32, 33 or 34 (1) 1-x.sub.1 73 x.sub.1 is
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 (1) 1-x.sub.1 75
x.sub.1 is 28, 29, 30, 31, 32, 33, 34, 35, 36, or 37 (1) 1-x.sub.1
77 x.sub.1 is 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34 (1)
1-x.sub.1 79 x.sub.1 is 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33
(1) 1-x.sub.1 81 x.sub.1 is 21, 22, 23, 24, 25, 26, 27, 28, 29 or
30 (1) 1-x.sub.1 83 x.sub.1 is 22, 23, 24, 25, 26, 27, 28, 29, 30
or 31 (1) 1-x.sub.1 85 x.sub.1 is 22, 23, 24, 25, 26, 27, 28, 29,
30 or 31 (1) 1-x.sub.1 87 x.sub.1 is 22, 23, 24, 25, 26, 27, 28,
29, 30 or 31 (1) 1-x.sub.1 89 x.sub.1 is 22, 23, 24, 25, 26, 27,
28, 29, 30 or 31 (1) 1-x.sub.1 91 x.sub.1 is 42, 43, 44, 45, 46,
47, 48, 49, 50 or 51 (1) 1-x.sub.1 93 x.sub.1 is 28, 29, 30, 31,
32, 33, 34, 35, 36 or 37 (1) 1-x.sub.1 95 x.sub.1 is 24, 25, 26,
27, 28, 29, 30, 31, 32 or 33 (1) 1-x.sub.1 97 x.sub.1 is 24, 25,
26, 27, 28, 29, 30, 31, 32 or 33 (1) x.sub.1 - x.sub.2 2 x.sub.1 is
25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 (2) x.sub.2 is 43, 44,
45, 46, 47, 48, 49, 50, 51, or 52 x.sub.1 - x.sub.2 5 x.sub.1 is
22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 (2) x.sub.2 is 41, 42,
43, 44, 45, 46, 47, 48, 49, 50 or 51 x.sub.1 - x.sub.2 43 x.sub.1
is 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 (2) x.sub.2 is 38, 39,
40, 41, 42, 43, 44, 45, 46 or 47 x.sub.1 - x.sub.2 45 x.sub.1 is
27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 (2) x.sub.2 is 46, 47, 48,
49, 50, 51, 52, 53, 54 or 55 x.sub.1 - x.sub.2 47 x.sub.1 is 43,
44, 44, 46 47, 48, 49, 50, 51 or 52 (2) x.sub.2 is 61, 62, 63, 64,
65, 66, 67, 68, 69 or 70 x.sub.1 - x.sub.2 49 x.sub.1 is 43, 44,
44, 46 47, 48, 49, 50, 51 or 52 (2) x.sub.2 is 61, 62, 63, 64, 65,
66, 67, 68, 69 or 70 x.sub.1 - x.sub.2 51 x.sub.1 is 50, 51, 52,
53, 54, 55, 56, 57, 58, 59 or 60 (2) x.sub.2 is 68, 69, 70, 71, 72,
73, 74, 75, 76, 77 or 78 x.sub.1 - x.sub.2 53 x.sub.1 is 50, 51,
52, 53, 54, 55, 56, 57, 58, 59 or 60 (2) x.sub.2 is 68, 69, 70, 71,
72, 73, 74, 75, 76, 77 or 78 x.sub.1 - x.sub.2 55 x.sub.1 is 19,
20, 21, 22, 23, 24, 25, 26 27, or 28 (2) x.sub.2 is 38, 39, 40, 41,
42, 43, 44, 45, 46 or 47 x.sub.1 - x.sub.2 57 x.sub.1 is 18, 19,
20, 21, 22, 23, 24, 25, 26 or 27 (2) x.sub.2 is 37, 38, 39, 40, 41,
42, 43, 44, 45 or 49 x.sub.1 - x.sub.2 59 x.sub.1 is 36, 37, 38,
29, 40, 41, 42, 43, 44 or 45 (2) x.sub.2 is 53, 54, 55, 56, 57, 58,
59, 60, 61 or 62 x.sub.1 - x.sub.2 61 x.sub.1 is 25, 26, 27, 28,
29, 30, 31, 32, 33 or 34 (2) x.sub.2 is 43, 44, 45, 46, 47, 48, 49,
50, 51, 52 or 53 x.sub.1 - x.sub.2 63 x.sub.1 is 25, 26, 27, 28,
29, 30, 31, 32, 33 or 34 (2) x.sub.2 is 43, 44, 45, 46, 47, 48, 49,
50, 51, 52 or 53 x.sub.1 - x.sub.2 65 x.sub.1 is 30, 31, 32, 33,
34, 35, 36, 37, 38, 39 or 40 (2) x.sub.2 is 48, 49, 50, 51, 52, 53,
54, 55, 56, 57 or 58 x.sub.1 - x.sub.2 67 x.sub.1 is 30, 31, 32,
33, 34, 35, 36, 37, 38, 39 or 40 (2) x.sub.2 is 48, 49, 50, 51, 52,
53, 54, 55, 56, 57 or 58 x.sub.1 - x.sub.2 69 x.sub.1 is 40, 41,
42, 43, 44, 45, 46, 47, 48 or 49 (2) x.sub.2 is 57, 58, 59, 60, 61,
62, 63, 64, 65 ot 66 x.sub.1 - x.sub.2 71 x.sub.1 is 25, 26, 27,
28, 29, 30, 31, 32, 33 or 34 (2) x.sub.2 is 43, 44, 45, 46, 47, 48,
49, 50, 51, or 52 x.sub.1 - x.sub.2 73 x.sub.1 is 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, or 55 (2) x.sub.2 is 63, 64, 65, 66, 67,
68, 69, 70, 71, 72 or 73 x.sub.1 - x.sub.2 75 x.sub.1 is 28, 29,
30, 31, 32, 33, 34, 35, 36, or 37 (2) x.sub.2 is 46, 47, 48, 49,
50, 51, 52, 53, 54 or 55 x.sub.1 - x.sub.2 77 x.sub.1 is 25, 26,
27, 28, 29, 30, 31, 32, 33 or 34 (2) x.sub.2 is 43, 44, 45, 46, 47,
48, 49, 50, 51, 52 or 53 x.sub.1 - x.sub.2 79 x.sub.1 is 24, 25,
26, 27, 28, 29, 30, 31, 32 or 33 (2) x.sub.2 is 44, 45, 46, 47, 48,
49, 50, 51, 52 or 53 x.sub.1 - x.sub.2 81 x.sub.1 is 21, 22, 23,
24, 25, 26, 27, 28, 29 or 30 (2) x.sub.2 is 39, 40, 41, 42, 43, 44,
45, 46, 47 or 48 x.sub.1 - x.sub.2 83 x.sub.1 is 22, 23, 24, 25,
26, 27, 28, 29, 30 or 31 (2) x.sub.2 is 41, 42, 43, 44, 45, 46, 47,
48, 49, 50 or 51 x.sub.1 - x.sub.2 85 x.sub.1 is 22, 23, 24, 25,
26, 27, 28, 29, 30 or 31 (2) x.sub.2 is 41, 42, 43, 44, 45, 46, 47,
48, 49, 50 or 51 x.sub.1 - x.sub.2 87 x.sub.1 is 22, 23, 24, 25,
26, 27, 28, 29, 30 or 31 (2) x.sub.2 is 41, 42, 43, 44, 45, 46, 47,
48, 49, 50 or 51 x.sub.1 - x.sub.2 89 x.sub.1 is 22, 23, 24, 25,
26, 27, 28, 29, 30 or 31 (2) x.sub.2 is 41, 42, 43, 44, 45, 46, 47,
48, 49, 50 or 51 x.sub.1 - x.sub.2 91 x.sub.1 is 42, 43, 44, 45,
46, 47, 48, 49, 50 or 51 (2) x.sub.2 is 59, 60, 61, 62, 63, 65, 65,
66, 67 or 68 x.sub.1 - x.sub.2 93 x.sub.1 is 28, 29, 30, 31, 32,
33, 34, 35, 36 or 37 (2) x.sub.2 is 46, 47, 48, 49, 50, 51, 52, 53,
54 or 55 x.sub.1 - x.sub.2 95 x.sub.1 is 24, 25, 26, 27, 28, 29,
30, 31, 32 or 33 (2) x.sub.2 is 44, 45, 46, 47, 48, 49, 50, 51, 52
or 53 x.sub.1 - x.sub.2 97 x.sub.1 is 24, 25, 26, 27, 28, 29, 30,
31, 32 or 33 (2) x.sub.2 is 44, 45, 46, 47, 48, 49, 50, 51, 52 or
53 x.sub.2 - x.sub.3 2 x.sub.2 is 43, 44, 45, 46, 47, 48, 49, 50,
51, or 52 (3) x.sub.3 is 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69 or 70 x.sub.2 - x.sub.3 5 x.sub.2 is 41, 42, 43, 44, 45, 46,
47, 48, 49, 50 or 51 (3) x.sub.3 is 55, 56, 57, 58, 59, 60, 61, 62,
63, 6, 4 65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 x.sub.2 -
x.sub.3 43 x.sub.2 is 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 (3)
x.sub.3 is 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65
x.sub.2 - x.sub.3 45 x.sub.2 is 46, 47, 48, 49, 50, 51, 52, 53, 54
or 55 (3) x.sub.3 is 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73 or 74 x.sub.2 - x.sub.3 47 x.sub.2 is 61, 62, 63, 64, 65,
66, 67, 68, 69 or 70 (3) x.sub.3 is 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87 or 88 x.sub.2 - x.sub.3 49 x.sub.2 is 61, 62,
63, 64, 65, 66, 67, 68, 69 or 70 (3) x.sub.3 is 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87 or 88 x.sub.2 - x.sub.3 51 x.sub.2
is 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 78 (3) x.sub.3 is 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95 x.sub.2 -
x.sub.3 53 x.sub.2 is 68, 69, 70, 71, 72, 73, 74, 75, 76, 77 or 78
(3) x.sub.3 is 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94
or 95 x.sub.2 - x.sub.3 55 x.sub.2 is 38, 39, 40, 41, 42, 43, 44,
45, 46 or 47 (3) x.sub.3 is 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64 or 65 x.sub.2 - x.sub.3 57 x.sub.2 is 37, 38, 39, 40,
41, 42, 43, 44, 45 or 49 (3) x.sub.3 is 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, or 64 x.sub.2 - x.sub.3 59 x.sub.2 is 53,
54, 55, 56, 57, 58, 59, 60, 61 or 62 (3) x.sub.3 is 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80 or 81 x.sub.2 - x.sub.3 61
x.sub.2 is 43, 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 (3) x.sub.3
is 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,
73, 74, 75 or 76 x.sub.2 - x.sub.3 63 x.sub.2 is 43, 44, 45, 46,
47, 48, 49, 50, 51, 52 or 53 (3) x.sub.3 is 58, 59, 60, 61, 62, 63,
64, 65, 66, 67, 68, 69 or 70 x.sub.2 - x.sub.3 65 x.sub.2 is 48,
49, 50, 51, 52, 53, 54, 55, 56, 57 or 58 (3) x.sub.3 is 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 x.sub.2 - x.sub.3 67
x.sub.2 is 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58 (3) x.sub.3
is 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 or 75 x.sub.2 -
x.sub.3 69 x.sub.2 is 57, 58, 59, 60, 61, 62, 63, 64, 65 ot 66 (3)
x.sub.3 is 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 or 84
x.sub.2 - x.sub.3 71 x.sub.2 is 43, 44, 45, 46, 47, 48, 49, 50, 51,
or 52 (3) x.sub.3 is 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64
or 65 x.sub.2 - x.sub.3 73 x.sub.2 is 63, 64, 65, 66, 67, 68, 69,
70, 71, 72 or 73 (3) x.sub.3 is 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89 or 90 x.sub.2 - x.sub.3 75 x.sub.2 is 46, 47, 48, 49,
50, 51, 52, 53, 54 or 55 (3) x.sub.3 is 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72 or 73 x.sub.2 - x.sub.3 77 x.sub.2 is 43, 44,
45, 46, 47, 48, 49, 50, 51, 52 or 53 (3) x.sub.3 is 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64 or 65 x.sub.2 - x.sub.3 79 x.sub.2 is
44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 (3) x.sub.3 is 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67 or 68 x.sub.2 - x.sub.3 81
x.sub.2 is 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 (3) x.sub.3 is
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66 x.sub.2 -
x.sub.3 83 x.sub.2 is 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51
(3) x.sub.3 is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, or 74 x.sub.2 - x.sub.3 85 x.sub.2 is 41,
42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 (3) x.sub.3 is 55, 56, 5,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, or
74 x.sub.2 - x.sub.3 87 x.sub.2 is 41, 42, 43, 44, 45, 46, 47, 48,
49, 50 or 51 (3) x.sub.3 is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, or 74 x.sub.2 - x.sub.3 89
x.sub.2 is 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or 51 (3) x.sub.3
is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, or 74 x.sub.2 - x.sub.3 91 x.sub.2 is 59, 60, 61, 62,
63, 65, 65, 66, 67 or 68 (3) x.sub.3 is 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86 or 87 x.sub.2 - x.sub.3 93 x.sub.2 is 46,
47, 48, 49, 50, 51, 52, 53, 54 or 55 (3) x.sub.3 is 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69 or 70 x.sub.2 - x.sub.3 95 x.sub.2
is 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 (3) x.sub.3 is 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 x.sub.2 - x.sub.3 97
x.sub.2 is 44, 45, 46, 47, 48, 49, 50, 51, 52 or 53 (3) x.sub.3 is
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 x.sub.3 - x.sub.4
2 x.sub.3 is 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70
(4) x.sub.4 is 78, 79, 80, 81, 82, 83, 84 or 85 x.sub.3 - x.sub.4 5
x.sub.3 is 55, 56, 57, 58, 59, 60, 61, 62, 63, 6, 4 65, 66, 67, 68,
(4) 69, 70, 71, 72, 73, or 74 x.sub.4 is 79, 80, 81, 82, 83, 84, 85
or 86 x.sub.3 - x.sub.4 43 x.sub.3 is 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64 or 65 (4) x.sub.4 is 73, 74, 75, 76, 77, 78,
79 or 80 x.sub.3 - x.sub.4 45 x.sub.3 is 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73 or 74 (4) x.sub.4 is 79, 80, 81, 82, 83,
84, 85 or 86 x.sub.3 - x.sub.4 47 x.sub.3 is 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87 or 88 (4) x.sub.4 is 95, 96, 97, 98,
99, 100, 101, 102 or 103 x.sub.3 - x.sub.4 49 x.sub.3 is 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 or 88 (4) x.sub.4 is 95,
96, 97, 98, 99, 100, 101, 102 or 103 x.sub.3 - x.sub.4 51 x.sub.3
is 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 or 95 (4)
x.sub.4 is 102, 103, 104, 105, 106, 107, 108, 109 or 110 x.sub.3 -
x.sub.4 53 x.sub.3 is 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,
93, 94 or 95 (4) x.sub.4 is 102, 103, 104, 105, 106, 107, 108, 109
or 110 x.sub.3 - x.sub.4 55 x.sub.3 is 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64 or 65 (4) x.sub.4 is 72, 73, 74, 75, 76, 77,
78, 79 or 80 x.sub.3 - x.sub.4 57 x.sub.3 is 51, 52, 53, 54, 55,
56, 57, 58, 59, 60, 61, 62, 63, or 64 (4) x.sub.4 is 71, 72, 73,
74, 75, 76, 77, 78 or 79 x.sub.3 - x.sub.4 59 x.sub.3 is 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or 81 (4) x.sub.4 is 78,
79, 80, 81, 82, 83, 84, 85 or 86 x.sub.3 - x.sub.4 61 x.sub.3 is
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, (4) 71, 72,
73, 74, 75 or 76 x.sub.4 is 82, 83, 84, 85, 86, 87, 88, 89 or
90
x.sub.3 - x.sub.4 63 x.sub.3 is 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69 or 70 (4) x.sub.4 is 77, 78, 79, 80, 81, 82, 83, 84 or
85 x.sub.3 - x.sub.4 65 x.sub.3 is 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74 or 75 (4) x.sub.4 is 83, 84, 85, 86, 87, 88, 89, 90
or 91 x.sub.3 - x.sub.4 67 x.sub.3 is 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74 or 75 (4) x.sub.4 is 83, 84, 85, 86, 87, 88, 89,
90 or 91 x.sub.3 - x.sub.4 69 x.sub.3 is 72, 73, 74, 75, 76, 77,
78, 79, 80, 81, 82, 83 or 84 (4) x.sub.4 is 92, 93, 94, 95, 96, 97,
98, 99 or 100 x.sub.3 - x.sub.4 71 x.sub.3 is 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64 or 65 (4) x.sub.4 is 72, 73, 74, 75, 76,
77, 78, 79 or 80 x.sub.3 - x.sub.4 73 x.sub.3 is 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89 or 90 (4) x.sub.4 is 98, 99, 100,
101, 102, 103, 104, 105 or 106 x.sub.3 - x.sub.4 75 x.sub.3 is 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 or 73 (4) x.sub.4 is 82,
83, 84, 85, 86, 87, 88, 89, or 90 x.sub.3 - x.sub.4 77 x.sub.3 is
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 or 65 (4) x.sub.4 is 72,
73, 74, 75, 76, 77, 78, 79 or 80 x.sub.3 - x.sub.4 79 x.sub.3 is
56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68 (4) x.sub.4 is
76, 77, 78, 79, 80, 81, 82, 83 or 84 x.sub.3 - x.sub.4 81 x.sub.3
is 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 or 66 (4) x.sub.4
is 74, 75, 76, 77, 78, 79, 80, 81 or 82 x.sub.3 - x.sub.4 83
x.sub.3 is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, (4) 70, 71, 72, 73, or 74 x.sub.4 is 79, 80, 81, 82, 83, 84,
85, 86 or 87 x.sub.3 - x.sub.4 85 x.sub.3 is 55, 56, 5, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, (4) 70, 71, 72, 73, or 74
x.sub.4 is 78, 79, 80, 81, 82, 83, 84, 85 or 86 x.sub.3 - x.sub.4
87 x.sub.3 is 55, 56, 5, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, (4) 70, 71, 72, 73, or 74 x.sub.4 is 79, 80, 81, 82, 83,
84, 85, 86 or 87 x.sub.3 - x.sub.4 89 x.sub.3 is 55, 56, 5, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, (4) 70, 71, 72, 73, or 74
x.sub.4 is 77, 78, 79, 80, 81, 82, 83, 84 or 85 x.sub.3 - x.sub.4
91 x.sub.3 is 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86 or
87 (4) x.sub.4 is 96, 97, 98, 99, 100, 101, 102, 103 or 104 x.sub.3
- x.sub.4 93 x.sub.3 is 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69 or 70 (4) x.sub.4 is 77, 78, 79, 80, 81, 82, 83, 84, or 85
x.sub.3 - x.sub.4 95 x.sub.3 is 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64 or 65 (4) x.sub.4 is 76, 77, 78, 79, 80, 81, 82, 83 or 84
x.sub.3 - x.sub.4 97 x.sub.3 is 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64 or 65 (4) x.sub.4 is 76, 77, 78, 79, 80, 81, 82, 83 or 84
x.sub.4 - x.sub.5 2 x.sub.4 is 78, 79, 80, 81, 82, 83, 84 or 85 (5)
x.sub.5 is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r 98 x.sub.4 -
x.sub.5 5 x.sub.4 is 79, 80, 81, 82, 83, 84, 85 or 86 (5) x.sub.5
is 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 x.sub.4 - x.sub.5 43
x.sub.4 is 73, 74, 75, 76, 77, 78, 79 or 80 (5) x.sub.5 is 85, 86,
87, 88, 89, 90 91, 92 or 93 x.sub.4 - x.sub.5 45 x.sub.4 is 79, 80,
81, 82, 83, 84, 85 or 86 (5) x.sub.5 is 91, 92, 93, 94, 95, 96, 97,
98, 99 or 100 x.sub.4 - x.sub.5 47 x.sub.4 is 95, 96, 97, 98, 99,
100, 101, 102 or 103 (5) x.sub.5 is 107, 108, 109, 110, 111, 112,
113, 114 or 115 x.sub.4 - x.sub.5 49 x.sub.4 is 95, 96, 97, 98, 99,
100, 101, 102 or 103 (5) x.sub.5 is 107, 108, 109, 110, 111, 112,
113, 114 or 115 x.sub.4 - x.sub.5 51 x.sub.4 is 102, 103, 104, 105,
106, 107, 108, 109 or 110 (5) x.sub.5 is 114, 115, 116, 117, 118,
119, 120, 121, or 122 x.sub.4 - x.sub.5 53 x.sub.4 is 102, 103,
104, 105, 106, 107, 108, 109 or 110 (5) x.sub.5 is 114, 115, 116,
117, 118, 119, 120, 121, or 122 x.sub.4 - x.sub.5 55 x.sub.4 is 72,
73, 74, 75, 76, 77, 78, 79 or 80 (5) x.sub.5 is 84, 85, 86, 87, 88,
89, 90, 91, 92 or 93 x.sub.4 - x.sub.5 57 x.sub.4 is 71, 72, 73,
74, 75, 76, 77, 78 or 79 (5) x.sub.5 is 83, 84, 85, 86, 87, 88, 89,
90, 91, or 92 x.sub.4 - x.sub.5 59 x.sub.4 is 78, 79, 80, 81, 82,
83, 84, 85 or 86 (5) x.sub.5 is 100, 101, 102, 103, 104, 106, 106,
107 or 108 x.sub.4 - x.sub.5 61 x.sub.4 is 82, 83, 84, 85, 86, 87,
88, 89 or 90 (5) x.sub.5 is 94, 95, 96, 97, 98, 99, 100, 101 or 102
x.sub.4 - x.sub.5 63 x.sub.4 is 77, 78, 79, 80, 81, 82, 83, 84 or
85 (5) x.sub.5 is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r 98 x.sub.4
- x.sub.5 65 x.sub.4 is 83, 84, 85, 86, 87, 88, 89, 90 or 91 (5)
x.sub.5 is 95, 96, 97, 98, 99, 100, 101, 102 or 103 x.sub.4 -
x.sub.5 67 x.sub.4 is 83, 84, 85, 86, 87, 88, 89, 90 or 91 (5)
x.sub.5 is 95, 96, 97, 98, 99, 100, 101, 102 or 103 x.sub.4 -
x.sub.5 69 x.sub.4 is 92, 93, 94, 95, 96, 97, 98, 99 or 100 (5)
x.sub.5 is 104, 105, 106, 107, 108, 109, 110, 111 or 112 x.sub.4 -
x.sub.5 71 x.sub.4 is 72, 73, 74, 75, 76, 77, 78, 79 or 80 (5)
x.sub.5 is 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 x.sub.4 -
x.sub.5 73 x.sub.4 is 98, 99, 100, 101, 102, 103, 104, 105 or 106
(5) x.sub.5 is 110, 111, 112, 113, 114, 115, 116, 117 or 118
x.sub.4 - x.sub.5 75 x.sub.4 is 82, 83, 84, 85, 86, 87, 88, 89, or
90 (5) x.sub.5 is 94, 95, 96, 97, 98, 99, 100, 101 or 102 x.sub.4 -
x.sub.5 77 x.sub.4 is 72, 73, 74, 75, 76, 77, 78, 79 or 80 (5)
x.sub.5 is 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 x.sub.4 -
x.sub.5 79 x.sub.4 is 76, 77, 78, 79, 80, 81, 82, 83 or 84 (5)
x.sub.5 is 88, 89, 90, 91, 92, 93, 94, 95 or 96 x.sub.4 - x.sub.5
81 x.sub.4 is 74, 75, 76, 77, 78, 79, 80, 81 or 82 (5) x.sub.5 is
86, 87, 88, 89, 90, 91, 92, 93 or 94 x.sub.4 - x.sub.5 83 x.sub.4
is 79, 80, 81, 82, 83, 84, 85, 86 or 87 (5) x.sub.5 is 91, 92, 93,
94, 95, 96, 97, 98, 99 or 100 x.sub.4 - x.sub.5 85 x.sub.4 is 78,
79, 80, 81, 82, 83, 84, 85 or 86 (5) x.sub.5 is 90, 91, 92, 93, 94,
95, 96, 97, 98 or 99 x.sub.4 - x.sub.5 87 x.sub.4 is 79, 80, 81,
82, 83, 84, 85, 86 or 87 (5) x.sub.5 is 91, 92, 93, 94, 95, 96, 97,
98, 99 or 100 x.sub.4 - x.sub.5 89 x.sub.4 is 77, 78, 79, 80, 81,
82, 83, 84 or 85 (5) x.sub.5 is 89, 90, 91, 92, 93, 94, 95, 96, 97
o r 98 x.sub.4 - x.sub.5 91 x.sub.4 is 96, 97, 98, 99, 100, 101,
102, 103 or 104 (5) x.sub.5 is 108, 109, 110, 111, 112, 113, 114,
115 or 116 x.sub.4 - x.sub.5 93 x.sub.4 is 77, 78, 79, 80, 81, 82,
83, 84, or 85 (5) x.sub.5 is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r
98 x.sub.4 - x.sub.5 95 x.sub.4 is 76, 77, 78, 79, 80, 81, 82, 83
or 84 (5) x.sub.5 is 88, 89, 90, 91, 92, 93, 94, 95 or 96 x.sub.4 -
x.sub.5 97 x.sub.4 is 76, 77, 78, 79, 80, 81, 82, 83 or 84 (5)
x.sub.5 is 88, 89, 90, 91, 92, 93, 94, 95 or 96 x.sub.5 - 114 2
x.sub.5 is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r 98 (6) x.sub.5 -
116 5 x.sub.5 is 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 (6)
x.sub.5 - 110 43 x.sub.5 is 85, 86, 87, 88, 89, 90 91, 92 or 93 (6)
x.sub.5 - 116 45 x.sub.5 is 91, 92, 93, 94, 95, 96, 97, 98, 99 or
100 (6) x.sub.5 - 132 47 x.sub.5 is 107, 108, 109, 110, 111, 112,
113, 114 or 115 (6) x.sub.5 - 132 49 x.sub.5 is 107, 108, 109, 110,
111, 112, 113, 114 or 115 (6) x.sub.5 - 139 51 x.sub.5 is 114, 115,
116, 117, 118, 119, 120, 121, or 122 (6) x.sub.5 - 139 53 x.sub.5
is 114, 115, 116, 117, 118, 119, 120, 121, or 122 (6) X.sub.5 - 110
55 x.sub.5 is 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 (6) x.sub.5
- 108 57 x.sub.5 is 83, 84, 85, 86, 87, 88, 89, 90, 91, or 92 (6)
x.sub.5 - 125 59 x.sub.5 is 100, 101, 102, 103, 104, 106, 106, 107
or 108 (6) x.sub.5 - 119 61 x.sub.5 is 94, 95, 96, 97, 98, 99, 100,
101 or 102 (6) x.sub.5 - 114 63 x.sub.5 is 89, 90, 91, 92, 93, 94,
95, 96, 97 o r 98 (6) x.sub.5 - 120 65 x.sub.5 is 95, 96, 97, 98,
99, 100, 101, 102 or 103 (6) x.sub.5 - 120 67 x.sub.5 is 95, 96,
97, 98, 99, 100, 101, 102 or 103 (6) x.sub.5 - 129 69 x.sub.5 is
104, 105, 106, 107, 108, 109, 110, 111 or 112 (6) x.sub.5 - 109 71
x.sub.5 is 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 (6) x.sub.5 -
135 73 x.sub.5 is 110, 111, 112, 113, 114, 115, 116, 117 or 118 (6)
x.sub.5 - 119 75 x.sub.5 is 94, 95, 96, 97, 98, 99, 100, 101 or 102
(6) x.sub.5 - 109 77 x.sub.5 is 84, 85, 86, 87, 88, 89, 90, 91, 92
or 93 (6) x.sub.5 - 113 79 x.sub.5 is 88, 89, 90, 91, 92, 93, 94,
95 or 96 (6) x.sub.5 - 110 81 x.sub.5 is 86, 87, 88, 89, 90, 91,
92, 93 or 94 (6) x.sub.5 - 116 83 x.sub.5 is 91, 92, 93, 94, 95,
96, 97, 98, 99 or 100 (6) x.sub.5 - 115 85 x.sub.5 is 90, 91, 92,
93, 94, 95, 96, 97, 98 or 99 (6) x.sub.5 - 116 87 x.sub.5 is 91,
92, 93, 94, 95, 96, 97, 98, 99 or 100 (6) x.sub.5 - 114 89 x.sub.5
is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r 98 (6) x.sub.5 - 134 91
x.sub.5 is 108, 109, 110, 111, 112, 113, 114, 115 or 116 (6)
x.sub.5 - 113 93 x.sub.5 is 89, 90, 91, 92, 93, 94, 95, 96, 97 o r
98 (6) x.sub.5 - 112 95 x.sub.5 is 88, 89, 90, 91, 92, 93, 94, 95
or 96 (6) x.sub.5 - 112 97 x.sub.5 is 88, 89, 90, 91, 92, 93, 94,
95 or 96 (6)
[0079] In some embodiments, domain 3 may be derived from the same
parent as either domain 2, domain 4 or both domain 3 and 4.
[0080] In some embodiment, J1 (Junction 1) between domain 1 and
domain 2 comprises the consensus sequence Z.sub.1Z.sub.2W, wherein
Z.sub.1 is selected from the group L, V, F, and M, and Z.sub.2 is G
or K, wherein 2 of the 3 amino acids are found at the C-terminus of
the first domain or the N-terminus of the second domain. In some
embodiment, J2 (Junction 2) between domain 2 and domain 3 comprises
the consensus sequence CZ.sub.1G, wherein Z.sub.1 is selected from
the group H, S, A, L, I, E, K, Q and D, wherein 2 of the 3 amino
acids are found at the C-terminus of the second domain or the
N-terminus of the third domain. In some embodiment, J3 (Junction 3)
between domain 3 and domain 4 comprises the consensus sequence
Z.sub.1Z.sub.2Z.sub.3, wherein Z.sub.1 is selected from the group
T, S, P, G and I, Z.sub.2 is selected from the group consisting of
N, K, V, M, H and Y, and Z.sub.3 is selected from the group
consisting of H, Y, S, T and P, wherein 2 of the 3 amino acids are
found at the C-terminus of the third domain or the N-terminus of
the fouth domain. In some embodiment, J4 (Junction 4) between
domain 4 and domain 5 comprises the consensus sequence
Z.sub.1CZ.sub.2, wherein Z.sub.1 is selected from the group C, S
and V, and Z.sub.2 is selected from the group consisting of V, A, I
and T, wherein 2 of the 3 amino acids are found at the C-terminus
of the fourth domain or the N-terminus of the Fifth domain. In some
embodiment, J5 (Junction 1) between the domain 5 and domain 6
comprises the consensus sequence Z.sub.1Z.sub.2Z.sub.3, wherein
Z.sub.1 is selected from the group L, R and V, Z.sub.2 is selected
from the group consisting of T, Q, Y, F and M, and Z.sub.3 is
selected from the group consisting of L, F, Y, K, I, Q, V and T,
wherein 2 of the 3 amino acids are found at the C-terminus of the
fifth domain or the N-terminus of the sixth domain.
[0081] In one embodiment, the disclosure provides the following
domains (Table B) for reach of the TGF-beta family members that may
be recombined to form a chimera of the disclosure having increased
or improved biological activity (e.g., resistance to inactivation
and the like).
TABLE-US-00002 TABLE B Do- Do- Do- Do- Do- Do- main 1 main 2 main 3
main 4 main 5 main 6 BMP-2 1-30 31-48 49-68 69-81 82-93 94-114
BMP-3 1-24 25-42 43-62 63-77 78-89 90-110 BMP-4 1-32 33-50 51-70
71-83 84-95 96-116 BMP-5 1-47 48-65 66-85 86-99 100-111 112-132
BMP-6 1-47 48-65 66-85 86-99 100-111 112-132 BMP-7 1-54 55-72 73-92
93-106 107-118 119-139 BMP-8 1-54 55-72 73-92 93-106 107-118
119-139 BMP-9 1-24 25-42 43-62 63-76 77-88 89-110 BMP-10 1-23 24-41
42-61 62-75 76-87 88-108 BMP-15 1-40 41-58 59-78 79-92 93-104
105-125 GDF-1 1-30 31-48 49-72 73-86 87-98 99-119 GDF-3 1-30 31-48
49-68 69-81 82-93 94-114 GDF-5 1-35 36-53 54-73 74-87 88-99 100-120
GDF-6 1-35 36-53 54-73 74-87 88-99 100-120 GDF-7 1-44 45-62 63-82
83-96 97-108 109-129 GDF-8 1-30 31-48 49-63 64-76 77-88 89-109
GDF-9 1-50 51-68 69-88 89-102 103-114 115-135 GDF-10 1-33 34-51
52-71 72-86 87-98 99-119 GDF-11 1-30 31-48 49-63 64-76 77-88 89-109
GDF-15 1-31 32-49 50-66 67-80 81-92 93-112 NODAL 1-26 27-44 45-64
65-78 79-90 91-110 ACTIVIN-A 1-27 28-45 46-68 69-83 84-95 96-116
Activin-B 1-27 28-45 46-68 69-82 83-94 95-115 Activin-C 1-27 28-45
46-68 69-83 84-95 96-116 Activin-E 1-27 28-45 46-68 69-81 82-93
94-114 INHIBIN-A 1-46 47-64 65-84 85-100 101-112 113-134 TGF-beta1
1-32 33-50 51-68 69-81 82-93 94-113 TGF-beta2 1-31 32-49 50-67
68-80 81-92 93-112 TGF-beta3 1-31 32-49 50-67 68-80 81-92
93-112
[0082] Thus, as illustrated by various embodiments herein, the
disclosure provides chimeric TGF-beta family polypeptides, wherein
a first TGF-beta family protein (i.e., a first parental protein) is
recombined with a second different TGF-beta family protein to
provide a chimeric polypeptide. Table 2, below, provides exemplary
chimeric polypeptides of the disclosure. In some embodiments, the
polypeptide comprises one or more domains of a BMP-2 protein,
wherein the segments of the BMP-2 protein comprise segment 1: amino
acid residue from about 1 to about x.sub.1 of SEQ ID NO:2 ("1b");
segment 2 is from about amino acid residue x.sub.1 to about x.sub.2
of SEQ ID NO:2 ("2b"); segment 3 is from about amino acid residue
x.sub.2 to about x.sub.3 of SEQ ID NO:2 ("3b"); segment 4 is from
about amino acid residue x.sub.3 to about x.sub.4 of SEQ ID NO:2
("4b"); segment 5 is from about amino acid residue x.sub.4 to about
x.sub.5 of SEQ ID NO:2 ("5b"); and segment 6 is from about amino
acid residue x.sub.5 to about x.sub.6 of SEQ ID NO:2 ("6b"); and
wherein: x.sub.1 is residue 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
or 35 of SEQ ID NO:2; x.sub.2 is residue 45, 46, 47, or 48 of SEQ
ID NO:2; x.sub.3 is residue 65, 66, 67, or 68 of SEQ ID NO:2;
x.sub.4 is residue 76, 77, 78, 79, 80, 81 or 82 of SEQ ID NO:2;
x.sub.5 is residue 88, 89, 90, 91, 92, 93, or 94 of SEQ ID NO:2;
and x.sub.6 is residue 112, 113, or 114 or SEQ ID NO:2,
corresponding to the C-terminus of BMP-2, such that a continguous
polypeptide comprising segments 1b2b3b4b5b6b comprises a wild-type
BMP-2 following the translation initiation codon (ATG). Homologs
and proteins having at least about 80%, 90%, 95%, 98%, and 99%
identity to the foregoing sequences are also included by the
disclosure.
[0083] In other embodiments, the polypeptide comprises one or more
domains of an activin protein, wherein the segments of the activin
protein comprise segment 1: amino acid residue from about 1 to
about x.sub.1 of SEQ ID NO:5 ("1a"); segment 2 is from about amino
acid residue x.sub.1 to about x.sub.2 of SEQ ID NO:5 ("2a");
segment 3 is from about amino acid residue x.sub.2 to about x.sub.3
of SEQ ID NO:5 ("3a"); segment 4 is from about amino acid residue
x.sub.3 to about x.sub.4 of SEQ ID NO:5 ("4a"); segment 5 is from
about amino acid residue x.sub.4 to about x.sub.5 of SEQ ID NO:5
("5a"); and segment 6 is from about amino acid residue x.sub.5 to
about x.sub.6 of SEQ ID NO: 5 ("6a"); and wherein: x.sub.1 is
residue 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 of SEQ ID
NO:5; x.sub.2 is residue 42, 43, 44, or 45 of SEQ ID NO:5; x.sub.3
is residue 61, 62, 63, or 64 of SEQ ID NO:5; x.sub.4 is residue 78,
79, 80, 81, 82, 83 or 84 of SEQ ID NO:5; x.sub.5 is residue 90, 91,
92, 93, 94, 95 or 96 of SEQ ID NO:5; and x.sub.6 is residue 114,
115, or 116 or SEQ ID NO:5, corresponding to the C-terminus of
activin, such that a continguous polypeptide comprising segments
1a2a3a4a5a6a comprises a wild-type mature activin protein. Homologs
and proteins having at least about 80%, 90%, 95%, 98%, and 99%
identity to the foregoing sequences are also included by the
disclosure.
[0084] In some embodiments, chimeric TGF-beta family polypeptide
has a chimeric segmental structure selected from the group
consisting of: 1b2b3b4b5b6b; 1b2b3b4b5b6a; 1b2b3b4b5a6a;
1b2b3b4b5a6b; 1b2b3a4a5a6a; 1b2b3a4a5b6a; 1b2a3a4a5a6a;
1b2a3a4a5a6a L66V/V67I; 1b(1a_II)2a3a4a5a6a; 1b2a3a4a5a6b;
1b2a3a4a5b6b; 1b2a3a4a5b6a; 1b2a3b4b5b6a; 1b2a3b4b5a6a; and
1b2a3b4b5a6b.
[0085] In other embodiment, the chimeric polypeptide may be fused
to an additional heterologous polypeptide to generate a chimeric
fusion polypeptide. The heterologous polypeptide may be, for
example, a peptide useful for purification or that permits
oligomerization of multiple chimeric polypeptides of the
disclosure. The heterologous may be chemically conjugated to the
chimeric polypeptide or may be operably linked in-frame with a
coding sequence for the chimeric polypeptide.
[0086] In more particular embodiments, the polypeptide comprises a
sequence that is (a) at least 80%, 90%, 95%, 98%, or 99% identical
to sequence selected from the group consisting of SEQ ID NO: 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 and has BMP-2
activity; (b) comprises a sequence as set forth in SEQ ID NO: 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33; (c) is encoded
by a polynucleotide comprising a sequence selected from the group
consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, and 32; or (d) comprises a sequence described by an
algorithm selected from the group consisting of 1b2b3b4b5b6b;
1b2b3b4b5b6a; 1b2b3b4b56a; 1b2b3b4b5a6b; 1b2b3b4a5a6a;
1b2b3b4a5b6b; 1b2b3b4a5a6b; 1b2b3b4a5b6a; 1b2b3a4a5a6a;
1b2b3a4a5a6b; 1b2b3a4a5b6b; 1b2b3a4a5b6a; 1b2b3a4b5b6b;
1b2b3a4b5b6a; 1b2b3a4b5a6a; 1b2b3a4b5a6b; 1b2a3a4a5a6a;
1b2a3a4a5a6b; 1b2a3a4a5b6b; 1b2a3a4a5b6a; 1b2a3a4b5b6b;
1b2a3a4b5b6a; 1b2a3a4b5a6b; 1b2a3a4b5a6a; 1b2a3b4b5b6b;
1b2a3b4b5b6a; 1b2a3b4b5a6a; 1b2a3b4b5a6b; 1b2a3b4a5a6a;
1b2a3b4a5b6a; 1b2a3b4a5b6b; 1b2a3b4a5a6b; 1b2a3a4a5a6a L66V/V67I;
and 1b(1a_II)2a3a4a5a6a. In yet another embodiment, the disclosure
provides a chimeric TGF-beta polypeptide comprising a segment from
BMP-2 and segments from BMP-7 (e.g., a 1b-BMP7 polypetpide; see,
e.g., SEQ ID NO:35). In yet another embodiment, the disclosure
provides a chimeric TGF-beta polypeptide comprising a segment from
BMP-2 and segments from BMP-9 (e.g., a 1b-BMP9; see, e.g., SEQ ID
NO:37). In yet another embodiment, the disclosure provides a
chimeric TGF-beta polypeptide comprising a segment from BMP-2 and
segments from GDF-7 (e.g., a 1b-GDF7; see, e.g., SEQ ID NO:39). In
yet another embodiment, the disclosure provides a chimeric TGF-beta
polypeptide comprising a segment from BMP-2 and segments from GDF-8
(e.g., a 1b-GDF8; see, e.g., SEQ ID NO:41). The chimeric
polypeptides of the disclosure retain a TGF-beta protein family
member activity. Such activity can be measured in any number of
ways as described below. In some embodiments, the chimeric
polypeptide has BMP-2 activity, but is not inhibited by Noggin.
[0087] In some embodiments, segment of a chimeric polypeptide is
100% identical to the parental strand from which the segment was
derived. In other embodiments the segment can comprise an amino
acid sequence that has at least 60%, 70%, 80%, 90%, 95%, 96%, 97%,
98%, or 99% or more identity to a corresponding segment in a
parental strand. For example, the segment may have one or more
conservative amino acid substitutions (e.g., 1-5 conservative amino
acid substitutions).
[0088] In some embodiments, the chimeric TGF-beta family
polypeptide may have improved activity compared to one or more of
the parental strands from which the chimeric polypeptide is
generated. Biological activity of a chimeric polypeptide of the
disclosure can be measured using any number of recognized assays in
the art for TGF-beta activity. Such assays include, but are not
limited to, BIAcore (Surface Plasmon Resonance); C.sub.2Cl.sub.2
luciferase assay: Smad 1/5 reporter system; HEK293 luciferase
assay: Smad 2/3 reporter system; FSH (Follicle Stimulating Hormone)
release assay: in rat pituitary cells; BRE (BMP Response Element)
luciferase assay: Smad 1/5 reporter HEK 293 cells; Cripto binding
assay: Luciferase response measured in presence/absence of Crptio;
Human Stem Cell assay: Maintenance or Differentiation of H9 cells;
NMR binding Studies; Micro mass culture: Bone formation measured in
Chick embryos; X-ray Crystallography: Determine Structure of
ligand:receptor complexes; Native Gel: Visualization of ligand:
receptor complexes; Size Exclusion Chromatography (SEC):
Visualization of ligand:receptor complexes; Velocity Scan
Ultracentrifugation: Visualize ligand:receptor complex formation;
and Seldi mass Spectrometry: Accurately determine size of
ligands.
[0089] The chimeric TGF-beta family polypeptides described herein
may be prepared in various forms, such as lysates, crude extracts,
or isolated preparations.
[0090] In some embodiments, the isolated chimeric polypeptide is a
substantially pure polypeptide composition. A "substantially pure
polypeptide" refers to a composition in which the polypeptide
species is the predominant species present (i.e., on a molar or
weight basis it is more abundant than any other individual
macromolecular species in the composition), and is generally a
substantially purified composition when the object species
comprises at least about 50 percent of the macromolecular species
present by mole or % weight. Generally, a substantially pure
polypeptide composition will comprise about 60% or more, about 70%
or more, about 80% or more, about 90% or more, about 95% or more,
and about 98% or more of all macromolecular species by mole or %
weight present in the composition. In some embodiments, the object
species is purified to essential homogeneity (i.e., contaminant
species cannot be detected in the composition by conventional
detection methods) wherein the composition consists essentially of
a single macromolecular species. Solvent species, small molecules
(<500 Daltons), and elemental ion species are not considered
macromolecular species.
[0091] In certain embodiments, the disclosure contemplates making
functional variants by modifying the structure of chimeras. Such
modifications may be made, for example, for such purposes as
enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf
life and resistance to proteolytic degradation in vivo, improve
stability, solubility, bioavailability, or biodistribution of the
chimeric protein, etc.). For example, but not by way of limitation,
the derivatives include chimeras that have been modified, e.g., by
acetylation, carboxylation, acylation glycosylation, pegylation,
phosphorylation, farnesylation, biotinylation, lipidation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to a cellular ligand or other protein
such as an organic deriatizing agent, etc. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to specific chemical cleavage, acetylation,
formylation, metabolic synthesis, etc. Additionally, the derivative
may contain one or more non-natural amino acids, such as those with
ketone-containing side chain, polyethylene glycols, lipids, poly-
or mono-saccharide, and phosphates. Effects of such non-natural
amino acid elements on the functionality of a chimeric TGF-beta
superfamily protein may be tested as described herein for other
TGF-beta superfamily protein variants. When a chimeric TGF-beta
superfamily protein is produced in cells by cleaving a nascent form
of the precursor protein, post-translational processing may also be
important for correct folding and/or function of the protein.
Different cells (such as CHO, HeLa, MDCK, 293, W138, NIH-3T3 or
HEK293) have specific cellular machinery and characteristic
mechanisms for such post-translational activities and may be chosen
to ensure the correct post-translational modification and
processing of the precursor protein into a chimeric TGF-beta
superfamily protein. In vitro cell-free expression system in
combination with its associated engineered tRNA synthase and tRNA
can be utilized to ensure the correct modification in a specific
amino acid position genetically tagged to introduce non-natural
amino acids.
[0092] Modified chimeras can also be produced, for instance, by
amino acid substitution, deletion, or addition. For instance, it is
reasonable to expect that an isolated replacement of a leucine with
an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid (e.g., conservative mutations) will
not have a major effect on the biological activity of the resulting
molecule. Conservative replacements are those that take place
within a family of amino acids that are related in their side
chains.
[0093] In certain embodiments, the disclosure contemplates making
mutations in a proteolytic cleavage site of the chimera sequence to
make the site less susceptible to proteolytic cleavage. Computer
analysis (using a commercially available software, e.g., MacVector,
Omega, PCGene, Molecular Simulation, Inc.) can be used to identify
proteolytic cleavage sites. As will be recognized by one of skill
in the art, most of the described mutations, variants or
modifications may be made at the nucleic acid level or, in some
cases, by post translational modification or chemical synthesis.
Such techniques are well known in the art.
[0094] In certain embodiments, the disclosure contemplates specific
mutations of a chimera sequences so as to alter the glycosylation
of the chimera. Such mutations may be selected so as to introduce
or eliminate one or more glycosylation sites, such as O-linked or
N-linked glycosylation sites. Asparagine-linked glycosylation
recognition sites generally comprise a tripeptide sequence,
asparagine-X-threonine (where "X" is any amino acid) which are
specifically recognized by appropriate cellular glycosylation
enzymes. The alteration may also be made by the addition of, or
substitution by, one or more serine or threonine residues to the
sequence of the wild-type polypeptide (for O-linked glycosylation
sites). A variety of amino acid substitutions or deletions at one
or both of the first or third amino acid positions of a
glycosylation recognition site (and/or amino acid deletion at the
second position) results in non-glycosylation at the modified
tripeptide sequence. Another means of increasing the number of
carbohydrate moieties is by chemical or enzymatic coupling of
glycosides to the polypeptide. Depending on the coupling mode used,
the sugar(s) may be attached to (a) arginine and histidine; (b)
free carboxyl groups; (c) free sulfhydryl groups such as those of
cysteine; (d) free hydroxyl groups such as those of serine,
threonine, or hydroxyproline; (e) aromatic residues such as those
of phenylalanine, tyrosine, or tryptophan; or (f) the amide group
of glutamine. These methods are described in WO 87/05330 published
Sep. 11, 1987, and in Aplin and Wriston (1981) CRC Crit. Rev.
Biochem., pp. 259-306, incorporated by reference herein. Removal of
one or more carbohydrate moieties present on a chimera may be
accomplished chemically and/or enzymatically. Chemical
deglycosylation may involve, for example, exposure to the compound
trifluoromethanesulfonic acid, or an equivalent compound. This
treatment results in the cleavage of most or all sugars except the
linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the amino acid sequence intact. Chemical deglycosylation is
further described by Hakimuddin et al. (1987) Arch. Biochem.
Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem. 118:131.
Enzymatic cleavage of carbohydrate moieties on polypeptides can be
achieved by the use of a variety of endo- and exo-glycosidases as
described by Thotakura et al. (1987) Meth. Enzymol. 138:350. The
nucleic acid and/or amino acid sequence of a propeptide may be
adjusted, as appropriate, depending on the type of expression
system used, as mammalian, yeast, insect and plant cells may all
introduce differing glycosylation patterns that can be affected by
the amino acid sequence of the peptide.
[0095] In some embodiments, the chimeric polypeptide can be in the
form of arrays. The polypeptide may be in a soluble form, for
example as solutions in the wells of mircotitre plates, or
immobilized onto a substrate. The substrate can be a solid
substrate or a porous substrate (e.g, membrane), which can be
composed of organic polymers such as polystyrene, polyethylene,
polypropylene, polyfluoroethylene, polyethyleneoxy, and
polyacrylamide, as well as co-polymers and grafts thereof. A solid
support can also be inorganic, such as glass, silica, controlled
pore glass (CPG), reverse phase silica or metal, such as gold or
platinum. The configuration of a substrate can be in the form of
beads, spheres, particles, granules, a gel, a membrane or a
surface. Surfaces can be planar, substantially planar, or
non-planar. Solid supports can be porous or non-porous, and can
have swelling or non-swelling characteristics. A solid support can
be configured in the form of a well, depression, or other
container, vessel, feature, or location. A plurality of supports
can be configured on an array at various locations, addressable for
robotic delivery of reagents, or by detection methods and/or
instruments.
[0096] The disclosure also provides polynucleotides encoding the
chimeric TGF-beta family polypeptides disclosed herein. The
polynucleotides may be operably linked to one or more heterologous
regulatory or control sequences that control gene expression to
create a recombinant polynucleotide capable of expressing the
polypeptide. Expression constructs containing a polynucleotide
encoding the chimeric polypeptide can be introduced into
appropriate host cells to express the polypeptide. Polynucleotide
sequences encoding various domains or full chimera of the
disclosure can be determined without undue efforts based upon the
various codons that are associated with an amino acid of in a
polypeptide. Furthermore, the disclosure provides exemplary
sequences of the TGF-.beta. family member. Deriving the sequences
of a domain or chimera from the sequences provided herein is
readily performed by one of skill in the art. Given the knowledge
of specific sequences of the TGF-beta family of proteins, and the
specific descriptions of the chimeric polypeptides herein (e.g.,
the segment structure of the chimeric domains), the nucleic acid
sequence of the engineered chimera will be apparent to the skilled
artisan. The knowledge of the codons corresponding to various amino
acids coupled with the knowledge of the amino acid sequence of the
polypeptides allows those skilled in the art to make different
polynucleotides encoding the polypeptides of the disclosure. Thus,
the present disclosure contemplates each and every possible
variation of the polynucleotides that could be made by selecting
combinations based on possible codon choices, and all such
variations are to be considered specifically disclosed for any of
the polypeptides described herein.
[0097] In some embodiments, the polynucleotides comprise
polynucleotides that encode the polypeptides described herein but
have about 80% or more sequence identity, about 85% or more
sequence identity, about 90% or more sequence identity, about 91%
or more sequence identity, about 92% or more sequence identity,
about 93% or more sequence identity, about 94% or more sequence
identity, about 95% or more sequence identity, about 96% or more
sequence identity, about 97% or more sequence identity, about 98%
or more sequence identity, or about 99% or more sequence identity
at the nucleotide level to a reference polynucleotide encoding a
chimera or parental TGF-beta family polypeptide.
[0098] In some embodiments, the isolated polynucleotides encoding
the polypeptides may be manipulated in a variety of ways to provide
for expression of the polypeptide. Manipulation of the isolated
polynucleotide prior to its insertion into a vector may be
desirable or necessary depending on the expression vector. The
techniques for modifying polynucleotides and nucleic acid sequences
utilizing recombinant DNA methods are well known in the art.
Guidance is provided in Sambrook et al., 2001, Molecular Cloning: A
Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press;
and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene
Pub. Associates, 1998, updates to 2007.
[0099] In some embodiments, the polynucleotides are operatively
linked to control sequences for the expression of the
polynucleotides and/or polypeptides. In some embodiments, the
control sequence may be an appropriate promoter sequence, which can
be obtained from genes encoding extracellular or intracellular
polypeptides, either homologous or heterologous to the host
cell.
[0100] In some embodiments, the control sequence may also be a
suitable transcription terminator sequence, a sequence recognized
by a host cell to terminate transcription. The terminator sequence
is operably linked to the 3' terminus of the nucleic acid sequence
encoding the polypeptide. Any terminator which is functional in the
host cell of choice may be used.
[0101] In some embodiments, the control sequence may also be a
suitable leader sequence, a nontranslated region of an mRNA that is
important for translation by the host cell. The leader sequence is
operably linked to the 5' terminus of the nucleic acid sequence
encoding the polypeptide. Any leader sequence that is functional in
the host cell of choice may be used.
[0102] In some embodiments, the control sequence may also be a
signal peptide coding region that codes for an amino acid sequence
linked to the amino terminus of a polypeptide and directs the
encoded polypeptide into the cell's secretory pathway. The 5' end
of the coding sequence of the nucleic acid sequence may inherently
contain a signal peptide coding region naturally linked in
translation reading frame with the segment of the coding region
that encodes the secreted polypeptide. Alternatively, the 5' end of
the coding sequence may contain a signal peptide coding region that
is foreign to the coding sequence. The foreign signal
peptide-coding region may be required where the coding sequence
does not naturally contain a signal peptide coding region.
[0103] The disclosure is further directed to a recombinant
expression vector comprising a polynucleotide encoding the chimeric
TGF-beta polypeptides described herein, and one or more expression
regulating regions such as a promoter and a terminator, a
replication origin, etc., depending on the type of hosts into which
they are to be introduced. In creating the expression vector, the
coding sequence is located in the vector so that the coding
sequence is operably linked with the appropriate control sequences
for expression.
[0104] The recombinant expression vector may be any vector (e.g., a
plasmid or virus), which can be conveniently subjected to
recombinant DNA procedures and can bring about the expression of
the polynucleotide sequence. The choice of the vector will
typically depend on the compatibility of the vector with the host
cell or in vitro cell-free reaction mixture into which the vector
is to be introduced. The vectors may be linear or closed circular
plasmids.
[0105] The expression vector may be an autonomously replicating
vector, i.e., a vector that exists as an extrachromosomal entity,
the replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the host cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. Furthermore, a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the host cell, or a transposon,
may be used.
[0106] In some embodiments, the expression vector of the disclosure
contains one or more selectable markers, which permit easy
selection of transformed cells. A selectable marker is a gene the
product of which provides for biocide or viral resistance,
resistance to heavy metals, prototrophy to auxotrophs, and the
like.
[0107] In another embodiments, the disclosure provides a host cell
comprising a polynucleotide encoding the chimeric TGF-beta
polypeptide, the polynucleotide being operatively linked to one or
more control sequences for expression of the fusion polypeptide in
the host cell. Host cells for use in expressing the fusion
polypeptides encoded by the expression vectors of the present
disclosure are well known in the art. Appropriate culture mediums
and growth conditions for the above-described host cells are well
known in the art.
[0108] Expression vectors can be designed for expression of
chimeras in prokaryotic or eukaryotic cells. For example, chimeras
of the disclosure can be expressed in bacterial or prokaryote cells
such as E. Coli, insect cells (e.g., the baculovirus expression
system), yeast cells, microalgae, plant cells or mammalian cells as
well as in vitro cell-free expression system. Some suitable host
cells are discussed further in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990).
[0109] While one example of an expression system discussed is an E.
coli expression system, to those skilled in the art, these proteins
can be easily be cloned into and expressed from a large number of
other expression systems. The advantages include, but are not
limited to, achieving post-translational modifications as would be
seen in the organism the protein was derived from (in this case H.
sapiens), expression of the ligands without the start methionine
required for bacterial expression, and easy incorporation of
non-natural amino acids or additional chemical modifications.
Suitable prokaryotes include but are not limited to eubacteria,
such as Gram-negative or Gram-positive organisms, for example,
Enterobacteriaceae such as E. coli. Various E. coli strains are
publicly available, such as E. coli K12 strain MM294 (ATCC 31,446);
E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27, 325)
and K5 772 (ATCC 53,635). In addition to prokaryotes, eukaryotic
microbes such as filamentous fungi or yeast are suitable cloning or
expression hosts for VEGF-E-encoding vectors. Saccharomyces
cerevisiae is a commonly used lower eukaryotic host
microorganism.
[0110] Suitable host cells for the expression of chimeras are
derived from unicellular and multicellular organisms. Examples of
invertebrate cells include insect cells such as Drosophila S2 and
Spodoptera Sf9, as well as plant cells. Plant expression systems
have also been used successfully to express modified proteins.
Examples of useful mammalian host cell lines include Chinese
hamster ovary (CHO) and COS cells. More specific examples include
monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651);
human embryonic kidney line (293 or 293 cells subcloned for growth
in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977));
Chinese hamster ovary cells/-DHFR(CHO, Urlaub and Chasin, Proc.
Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,
Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138,
ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse
mammary tumor (MMT 060562, ATCC CCL51). The selection of the
appropriate host cell is deemed to be within the skill in the
art.
[0111] Alternate protein expression systems include human embryonic
kidney (HEK) 293 cells, insect cell line (S. frugiperda) utilizing
the baculovirus expression system, yeast expression systems not
limited to P. pastoris and S. cerevisiae, and numerous Microalgae
strains. Transgenic animals can be used to express correctly
modified protein. In essence, the animals become living
`bioreactors` capable of expressing large amounts of the desired
protein in an easily harvested fluid or tissue, such as the milk
from a cow. Cell-free in vitro expression systems using either the
bacterial or wheat germ cell lysate can be employed. Cell-free
expression system will permit inserting a wide range of non-natural
amino acids or epitope tags with higher efficiency and greater
specificity.
[0112] Examples of bacterial vectors include pQE70, pQE60, pQE-9
(Qiagen), pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks,
pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3,
pKK233-3, pDR540, and pRIT5 (Pharmacia). Examples of vectors for
expression in the yeast S. cerevisiae include pYepSec1 (Baldari et
al., EMBO J. 6:229 (1987)), pMFa (Kurjan and Herskowitz, Cell
30:933 (1982)), pJRY88 (Schultz et al., Gene 54:113 (1987)), and
pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirus
vectors available for expression of nucleic acids to produce
proteins in cultured insect cells (e.g., Sf9 cells) include the pAc
series (Smith et al., Mol. Cell. Biol. 3:2156 (1983)) and the pVL
series (Lucklow and Summers Virology 170:31 (1989)).
[0113] Examples of mammalian expression vectors include pWLNEO,
pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, PBPV, pMSG, PSVL
(Pharmacia), pCDM8 (Seed, Nature 329:840 (1987)) and pMT2PC
(Kaufman et al., EMBO J. 6:187 (1987)). When used in mammalian
cells, the expression vector's control functions are often provided
by viral regulatory elements. For example, commonly used promoters
are derived from polyoma, adenovirus 2, cytomegalovirus and Simian
Virus 40.
[0114] Viral vectors have been used in a wide variety of gene
delivery applications in cells, as well as living animal subjects.
Viral vectors that can be used include, but are not limited to,
retrovirus, lentivirus, adeno-associated virus, poxvirus,
alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr
virus, adenovirus, geminivirus, and caulimovirus vectors. Non-viral
vectors include plasmids, liposomes, electrically charged lipids
(cytofectins), nucleic acid-protein complexes, and biopolymers. In
addition to a nucleic acid of interest, a vector may also comprise
one or more regulatory regions, and/or selectable markers useful in
selecting, measuring, and monitoring nucleic acid transfer results
(delivery to specific tissues, duration of expression, etc.).
[0115] The chimera of the disclosure can be made by using methods
well known in the art. Polynucleotides can be synthesized by
recombinant techniques, such as that provided in Sambrook et al.,
2001, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring
Harbor Laboratory Press; and Current Protocols in Molecular
Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to
2007. Polynucleotides encoding the enzymes, or the primers for
amplification can also be prepared by standard solid-phase methods,
according to known synthetic methods, for example using
phosphoramidite method described by Beaucage et al., (1981) Tet
Lett 22:1859-69, or the method described by Matthes et al., (1984)
EMBO J. 3:801-05, e.g., as it is typically practiced in automated
synthetic methods. In addition, automated peptide synthesizers are
commercially available (e.g., Advanced ChemTech Model 396;
Milligen/Biosearch 9600).
[0116] In a one embodiment, the disclosure is directed to a method
to accelerate construction of large chimera libraries. Accordingly,
the disclosure provides a recombinant strategy termed RASCH (RAndom
Segmental CHimera) See FIG. 17. It uses a template sequence (first
strand from one TGF-beta superfamily member) and a few target
sequences (second (third, fourth, fifth, sixth) strand from one or
more alternate TGF-beta superfamily members), whose subdomains are
to be linked. The template DNA sequence is used to promote
efficient coupling of the target sequences and is degraded once
subdomains are linked. Following the gene construction to create
the chimeric sequences, the new ligands are chemically refolded
into functional dimer. This dimerization process permits additional
diversification of the final sequence by mixing and dimerizing two
different sequences of both natural and designer origins.
Therefore, the RASCH method can be used to diversify the
approximate 40 natural protein sequences of TGF-beta superfamily
ligands into ten of thousands or more variant sequences, each
distinct from any naturally-ocurring TGF-beta superfamily ligand
sequences.
[0117] Engineered polypeptide expressed in a host cell can be
recovered from the cells and or the culture medium using any one or
more of the well known techniques for protein purification,
including, among others, lysozyme treatment, sonication,
filtration, salting-out, ultra-centrifugation, chromatography, and
affinity separation (e.g., substrate bound antibodies).
[0118] Chromatographic techniques for isolation of the polypeptides
include, among others, reversed phase chromatography high
performance liquid chromatography, ion exchange chromatography, gel
electrophoresis, and affinity chromatography. Conditions for
purifying a particular enzyme will depend, in part, on factors such
as net charge, hydrophobicity, hydrophilicity, molecular weight,
molecular shape, etc., and will be apparent to those having skill
in the art.
[0119] Assays to determine activity are well known in the art. The
present disclosure relates to assays to test for biological
activity of chimeric proteins, more preferably, to assays to test
for clinical activity. Such activity can include enhanced agonistic
or antagonistic TGF-beta activity, combined or novel biological
activity, and the like.
[0120] In certain embodiments, a chimeric protein of the disclosure
comprising an agonist of a TGF-beta superfamily protein comprises
an antagonist of a different TGF-beta superfamily protein.
[0121] Irrespective of which protein expression, harvesting, and,
folding methodologies are used, certain of the subject chimeric
proteins can bind, preferentially to a pre-selected receptor and
can now be identified using standard methodologies, e.g.,
ligand/receptor binding assays, well known, and thoroughly
documented in the art. See, e.g., Legerski gl al. (1992) Bio
h.sup.-_Biophys. Res. Comm. 183: 672679; Frakar et al. (1978)
Biochem. Bio12-hys. Res. Comm 80:849-857; Chio et el. (1990) Nature
343: 266-269; Dahlman et al. (1988) Biochem 27: 1813-1817; Strader
et el. (1989) J. Biol. Chem. 264: 13572-13578; and DDowd et al.
(1988) J. Biol. Chem. 263: 15985-15992.
[0122] Typically, in a ligand/receptor binding assay, the native or
parent TGF-beta superfamily member of interest having a known,
quantifiable affinity for a pre-selected receptor is labeled with a
detectable moiety, for example, a radiolabel, a chromogenic label,
or a fluorogenic label. Aliquots of purified receptor, receptor
binding domain fragments, or cells expressing the receptor of
interest on their surface are incubated with the labeled TGF-beta
superfamily member in the presence of various concentrations of the
unlabeled chimeric protein. The relative binding affinity of a
candidate chimeric protein may be measured by quantitating the
ability of the chimeric protein to inhibit the binding of the
labeled TGF-beta superfamily member with the receptor. In
performing the assay, fixed concentrations of the receptor and the
TGF-beta superfamily member are incubated in the presence and
absence of unlabeled chimeric protein. Sensitivity may be increased
by preincubating the receptor with the chimeric protein before
adding the labeled template TGF-beta superfamily member. After the
labeled competitor has been added, sufficient time is allowed for
adequate competitor binding, and then free and bound labeled
TGF-beta superfamily members are separated from one another, and
one or the other measured. Labels useful in the practice of the
screening procedures include radioactive labels, chromogenic
labels, spectroscopic labels such as those disclosed in Haughland
(1994) "Handbook of Fluorescent and Research Chemicals," 5 ed. by
Molecular Probes, Inc., Eugene, Oreg., or conjugated enzymes having
high turnover rates, i.e., horseradish peroxidase, alkaline
phosphatase, or agalactosidase, used in combination with
chemiluminescent or fluorogenic substrates. The biological
activity, namely the agonist or antagonist properties of the
resulting chimeric protein constructs can subsequently be
characterized using conventional in vivo and in vitro assays that
have been developed to measure the biological activity of any
TGF-beta superfamily member. It is appreciated, however, the type
of assay used preferably depends on the TGF-a superfamily member
upon which the chimeric protein is based. For example, chimeric
constructs based upon naturally occurring BMP-2 protein may be
assayed using any of the biological assays that have been developed
to date for measuring BMP-2 activity, described in more detail
below.
[0123] The presence of multimers among the subject chimeric
proteins can be detected visually either by standard SDS-PAGE in
the absence of a reducing agent such as DTT or by HPLC (e.g., C18
reverse phase HPLC). Multimeric proteins of the present disclosure
can have an apparent molecular weight proportionally greater than
the monomeric subunit, e.g., in the range about 28-36 kDa for a
dimer, as compared to monomeric subunits, which may have an
apparent molecular weight of about 14-18 kDa. The multimeric
protein can readily be visualized on an electrophoresis gel by
comparison to commercially available molecular weight standards.
The dimeric protein also elutes from a C18 RP HPLC (45-50%
acetonitrile: 0.1% TFA) at a time point different from that for its
monomeric counterpart.
[0124] A second assay evaluates the presence of dimer (e.g., OP-1
dimers) by its ability to bind to hydroxyapatite. Optimally-folded
dimer binds a hydroxyapatite column well in pH7, 10 mM phosphate,
6M urea, and 0.142M NaCl (dimer elutes at 0.25 M NaCl) as compared
to monomer, which does not bind substantially at those
concentrations (monomer elutes at 0.1M NaCl). A third assay
evaluates the presence of dimer by the protein's resistant to
trypsin or pepsin digestion. The folded dimeric species is
substantially resistant to both enzymes, particularly trypsin,
which cleaves only a small portion of the N-terminus of the mature
protein, leaving a biologically active dimeric species only
slightly smaller in size than the untreated dimer (each monomer in
amino acids smaller after trypsin cleavage). By contrast, the
monomers and misfolded dimers are substantially degraded. In the
assay, the protein is subjected to an enzyme digest using standard
conditions, e.g., digestion in a standard buffer such as 50 mM Tris
buffer, pH 8, containing 4M urea, 100 mM NaCl, 0.3% Tween-80 and 20
mM methylamine. Digestion is allowed to occur at 37.degree. C. for
on the order of 16 hours, and the product visualized by any
suitable means, preferably SDS PAGE.
[0125] The biological activity of the subject chimeric proteins,
for example, the chimeric proteins having one or more segments from
BMPs, can be assessed by any of a number of means as described in
WO00/20607. For example, the protein's ability to induce
endochondral bone formation can be evaluated using the well
characterized rat subcutaneous bone assay. In the assay bone
formation is measured by histology, as well as by alkaline
phosphatase and/or osteoclacin production. In addition, osteogenic
proteins having high specific bone forming activity, such as OP-1,
BMP-2, BMR4, BMP-5 and BMP-6, also induce alkaline phosphatase
activity in an in vitro rat osteoblast or osteosarcoma cell-based
assay. Such assays are well described in the art. See, for example,
Sabokdar of al. (1994) Bone and Mineral 27:57-67.; Knutsen et al.
(1993) Biochem Biophvs Res. Commun 194:1352-1358; and Maliakal et
al. (1994) Growth Factors 1:227-234).
[0126] By contrast, osteogenic proteins having low specific bone
forming activity, such as CDMP-1 and CDMP-2, for example, do not
induce alkaline phosphatase activity in the cell based osteoblast
assay. The assay thus provides a ready method for evaluating
biological activity of B1b9 mutants. For example, CDMP-1, CDMP-2
and CMDP-3 all are competent to induce bone formation, although
with a lower specific activity than BMP-2, BW-4, BV-5, BMP-6 or
OP-1. Conversely, BMP-2, BMP-4, BMP-5, BPylP-6 and OP-1 all can
induce articular cartilage formation, albeit with a lower specific
activity than CDMP-1, CDMP-2 or CDMP-3. Accordingly, a chimeric
protein having one or more segment from CDMP, designed and
described herein to be a chimeric protein competent to induce
alkaline phosphatase activity in the cell-based assay, is expected
to demonstrate a higher specific bone forming activity in the rat
animal bioassay.
[0127] The chimeric protein's biological activity can also be
readily evaluated by the protein's ability to inhibit epithelial
cell growth. A useful, well characterized in vitro assay utilizes
mink lung cells or melanoma cells. See WO00/20607. Other assays for
other members of the TGF-beta superfamily are well described in the
literature and can be performed without undue experimentation.
[0128] In certain embodiment, the disclosure provides methods and
agents for control and maintain skeletal muscle mass in a host,
preferably a human. Therefore, any chimeric protein of the
disclosure that is expected to affect muscle-related function of a
TGF-beta superfamily protein such as for example GDF-8 can be
tested in whole cells or tissues, in vitro or in vivo, to confirm
their ability to modulate skeletal muscle mass. GDF-8 (also known
as myostatin) is a negative regulator of skeletal muscle growth.
GDF-8 knockout mice have approximately twice the skeletal muscle
mass of normal mice. The effects of increased muscle mass on bone
modeling may be investigated, e.g., by examining bone mineral
content (BMC) and bone mineral density (BMD) in the femora of
female GDF-8 knockout mice. Dual-energy X-ray absorptiometry (DEXA)
densitometry can be used to measure whole-femur BMC and BMD, and
PQCT densitometry can be used to calculate BMC and BMD from
cross-sections of tissues. Hamrick, Anat Rec. 2003 May;
272A(1):388-91. As is known in the art, a chimeric protein of the
disclosure may be introduced into the GDF-8 knockout mice, and
similar assays can be used to determine the effect of the chimeric
protein on skeletal muscle mass and bone density.
[0129] The dystrophic phenotype in the mdx mouse model of Duchenne
muscular dystrophy (DMD) may also be employed to test the
biological activity of a chimeric protein of the disclosure. It was
reported that blockade of endogenous myostatin by using
intraperitoneal injections of blocking antibodies for three months
resulted in an increase in body weight, muscle mass, muscle size
and absolute muscle strength in mdx mouse muscle along with a
significant decrease in muscle degeneration and concentrations of
serum creatine kinase. Bogdanovich et al., Nature. 2002 Nov. 28;
420(6914):418-21. Similar study may be employed to determine
whether a chimeric protein of the disclosure potentiates or
inhibits the endogenous GDF-8 activity.
[0130] In certain embodiments, the disclosure provides methods and
agents for modulating neurogenesis. For example, GDF-11 is known to
inhibit olfactory epithelium neurogenesis in vitro by inducing p27
(Kip1) and reversible cell cycle arrest in progenitors. Wu et al.
Neuron. 2003 Jan. 23; 37(2):197-207. The effect of a chimeric
protein of the disclosure on neurogenesis can be similarly tested.
Further, the effect of a chimeric protein of the disclosure on
GDF-11's effect on neurogenesis can also be tested using similar
assays as described in Wu et al. Id.
[0131] In certain embodiment, the disclosure provides methods and
agents for stimulating bone formation and increasing bone mass.
Therefore, any chimeric protein of the disclosure that is expected
to affect bone-related function of a TGF-beta superfamily protein
such as for example BMP-2, BMP-3, GDF-10, BMP-4, BMP-7, or BMP-8,
can be tested in whole cells or tissues, in vitro or in vivo, to
confirm their ability to modulate bone or cartilage growth. Various
methods known in the art can be utilized for this purpose.
For example, BMP-3 inhibits BMP2-mediated induction of Msx2 and
blocks BMP2-mediated differentiation of osteoprogenitor cells into
osteoblasts. Thus, the effect of a subject chimer protein,
preferably one comprising a segment from a BMP-2 or BMP-3, on bone
or cartilage growth can be determined by their effect on the
osteogenic activity of BMP-2, for example, by measuring induction
of Msx2 or differentiation of osteoprogenitor cells into
osteoblasts in cell based assays (see, e.g., Daluiski et al., Nat.
Genet. 2001, 27(1):84-8; Hino et al., Front Biosci. 2004,
9:1520-9). Similarly, a subject chimeric protein, preferably one
comprising a segment from a BMP-2 or BMP-3, may be tested for its
osteogenic or anti-osteogenic activity or its agonistic or
antagonistic effect on BMP-2-mediated osteogenesis.
[0132] Another example of cell-based assays includes analyzing the
osteogenic or anti-osteogenic activity of a subject chimeric and
test compounds in mesenchymal progenitor and osteoblastic cells. To
illustrate, recombinant adenoviruses expressing a subject chimeric
protein were constructed to infect pluripotent mesenchyimal
progenitor C3H10T1/2 cells, preosteoblastic C2C12 cells, and
osteoblastic TE-85 cells. Osteogenic activity is then determined by
measuring the induction of alkaline phosphatase, osteocalcin, and
matrix mineralization (see, e.g., Cheng et al., J bone Joint Surg
Am. 2003, 85-A(8):1544-52).
[0133] Further, the disclosure contemplates in vivo assays to
measure bone or cartilage growth. For example, Namkung-Matthai et
al., Bone, 28:80-86 (2001) discloses a rat osteoporotic model in
which bone repair during the early period after fracture is
studied. Kubo et al., Steroid Biochemistry & Molecular Biology,
68:197-202 (1999) also discloses a rat osteoporotic model in which
bone repair during the late period after fracture is studied. These
references are incorporated by reference herein in their entirety
for their disclosure of rat model for study on osteoporotic bone
fracture. In certain aspects, the present disclosure makes use of
fracture healing assays that are known in the art. These assays
include fracture technique, histological analysis, and
biomechanical analysis, which are described in, for example, U.S.
Pat. No. 6,521,750, which is incorporated by reference in its
entirety for its disclosure of experimental protocols for causing
as well as measuring the extent of fractures, and the repair
process.
[0134] It is understood that the screening assays of the disclosure
apply to not only the subject chimeric proteins and variants
thereof, but also any test compounds including agonists and
antagonist of the chimeric proteins or their variants themselves.
Further, these screening assays are useful for drug target
verification and quality control purposes.
[0135] In other embodiment, the disclosure relates to the use of
the subject chimeric TGF-beta superfamily proteins to identify
compounds which can modulate activities of the chimeric proteins.
Compounds identified through this screening can be tested in
tissues (e.g., bone and/or cartilage) or cells (e.g., muscle cells)
to assess their ability to modulate the test tissues or cells
(e.g., bone/cartilage growth or muscle cell growth) in vitro.
Optionally, these compounds can further be tested in animal models
to assess their ability to modulate, e.g., bone/cartilage growth or
muscle control and maintenance in vivo.
[0136] A variety of assay formats will suffice and, in light of the
disclosure, those not expressly described herein will nevertheless
be comprehended by one of ordinary skill in the art. As described
herein, the test compounds (agents) of the disclosure may be
created by any combinatorial chemical method. Alternatively, the
subject compounds may be naturally occurring biomolecules
synthesized in vivo or in vitro. Compounds (agents) to be tested
for their ability to act as modulators of bone or cartilage growth
can be produced, for example, by bacteria, yeast, plants or other
organisms (e.g., natural products), produced chemically (e.g.,
small molecules, including peptidomimetics), or produced
recombinantly. Test compounds contemplated by the present
disclosure include non-peptidyl organic molecules, peptides,
polypeptides, peptidomimetics, sugars, hormones, and nucleic acid
molecules. In a specific embodiment, the test agent is a small
organic molecule having a molecular weight of less than about 2,000
daltons.
[0137] The test compounds of the disclosure can be provided as
single, discrete entities, or provided in libraries of greater
complexity, such as made by combinatorial chemistry. These
libraries can comprise, for example, alcohols, alkyl halides,
amines, amides, esters, aldehydes, ethers and other classes of
organic compounds. Presentation of test compounds to the test
system can be in either an isolated form or as mixtures of
compounds, especially in initial screening steps. Optionally, the
compounds may be optionally derivatized with other compounds and
have derivatizing groups that facilitate isolation of the
compounds. Non-limiting examples of derivatizing groups include
biotin, fluorescein, digoxygenin, green fluorescent protein,
isotopes, polyhistidine, magnetic beads, glutathione S transferase,
photoactivatible crosslinkers or any combinations thereof.
[0138] In many drug screening programs which test libraries of
compounds and natural extracts, high throughput assays are
desirable in order to maximize the number of compounds surveyed in
a given period of time. Assays which are performed in cell-free
systems, such as may be derived with purified or semi-purified
proteins, are often preferred as "primary" screens in that they can
be generated to permit rapid development and relatively easy
detection of an alteration in a molecular target which is mediated
by a test compound. Moreover, the effects of cellular toxicity or
bioavailability of the test compound can be generally ignored in
the in vitro system, the assay instead being focused primarily on
the effect of the drug on the molecular target as may be manifest
in an alteration of binding affinity between a chimeric TGF-beta
superfamily protein and its binding protein (e.g., the chimeric
protein itself or a TGF-beta receptor protein or fragments
thereof).
[0139] Merely to illustrate, in an exemplary screening assay of the
present disclosure, the compound of interest is contacted with an
isolated and purified chimeric protein which is ordinarily capable
of binding to a TGF-beta receptor protein or fragments thereof, as
appropriate for the intention of the assay. To the mixture
comprising a subject chimeric protein and a TGF-beta receptor
protein is then added a composition containing a test compound.
Detection and quantification of the chimeric protein receptor
complexes provides a means for determining the compound's efficacy
at inhibiting (or potentiating) complex formation between the
chimeric TGF-beta superfamily protein and its binding protein,
e.g., the TGF-beta receptor or fragments thereof. The efficacy of
the compound can be assessed by generating dose response curves
from data obtained using various concentrations of the test
compound. Moreover, a control assay can also be performed to
provide a baseline for comparison. For example, in a control assay,
an isolated and purified chimeric TGF-beta superfamily protein is
added to a composition (cell-free or cell-based) containing a
TGF-beta receptor protein or fragment thereof, and the formation of
the chimeric protein-receptor complex is quantitated in the absence
of the test compound. It will be understood that, in general, the
order in which the reactants may be admixed can be varied, and can
be admixed simultaneously. Moreover, in place of purified proteins,
cellular extracts and lysates may be used to render a suitable
cell-free assay system. Alternatively, cells expressing a TGF-beta
receptor protein or fragments thereof on their surfaces can be used
in certain assays.
[0140] Complex formation between a subject chimeric TGF-beta
superfamily protein and its binding protein may be detected by a
variety of techniques. For instance, modulation of the formation of
complexes can be quantitated using, for example, detectably labeled
proteins such as radiolabelled (e.g., 32 P, 35 S, 14 C or 3 H),
fluorescently labeled (e.g., FITC), or enzymatically labeled
chimeric protein or its binding protein, by immunoassay, or by
chromatographic detection.
[0141] In certain embodiments, the present disclosure contemplates
the use of fluorescence polarization assays and fluorescence
resonance energy transfer (FRET) assays in measuring, either
directly or indirectly, the degree of interaction between a
chimeric TGF-beta superfamily protein and its binding protein
(e.g., a TGF-beta receptor protein or fragments thereof). Further,
other modes of detection such as those based on optical waveguides
(PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196), surface
plasmon resonance (SPR), surface charge sensors, and surface force
sensors are compatible with many embodiments of the disclosure.
[0142] Moreover, the present disclosure contemplates the use of an
interaction trap assay, also known as the "two hybrid assay," for
identifying agents that disrupt or potentiate interaction between a
chimeric TGF-beta superfamily protein and its binding protein
(e.g., a TGF-beta receptor protein or fragments thereof). See for
example, U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell
72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054;
Bartel et al. (1993) Biotechniques 14:920-924; and Iwabuchi et al.
(1993) Oncogene 8:1693-1696).
[0143] Chimera polynucleotides, polypeptides, antibodies, cells and
other reagents of the disclosure have a wide variety of uses, both
in vitro and in vivo. For example, in representative embodiments,
these reagents may be used in vitro or in vivo (e.g., in an animal
model) to study the processes of mineralization, bone formation,
and bone loss. Further, "knock in" and "knock out" animals can be
used as animal models of disease or as screening tools (discussed
more below) for compounds that interact with the chimera
polynucleotides or polypeptides. It will be apparent to those
skilled in the art that any suitable vector can be used to deliver
the polynucleotide to a cell or subject. The choice of delivery
vector can be made based on a number of factors known in the art,
including age and species of the target host, in vitro versus in
vivo delivery, level and persistence of expression desired,
intended purpose (e.g., for therapy or screening), the target cell
or organ, route of delivery, size of the isolated polynucleotide,
safety concerns, and the like.
[0144] Chimeric polypeptide of the disclosure may be formulated for
use in various biological systems including in vivo. Any of a
variety of art-known methods can be used to administer a chimera
either alone or in combination with other active agents. For
example, administration can be parenterally by injection or by
gradual infusion over time. The agent(s) can be administered by
such means as oral, rectal, buccal (e.g., sub-lingual), vaginal,
parenteral (e.g., subcutaneous, intramuscular including skeletal
muscle, cardiac muscle, diaphragm muscle and smooth muscle,
intradermal, intravenous, intraperitoneal), topical (i.e., both
skin and mucosal surfaces, including airway surfaces), intranasal,
transdermal, intraarticular, intrathecal, intracavity, and
inhalation administration, administration to the liver by
intraportal delivery, as well as direct organ injection (e.g., into
the liver, into the brain for delivery to the central nervous
system, into the pancreas). The most suitable route in any given
case will depend on the nature and severity of the condition being
treated and on the nature of the particular compound which is being
used.
[0145] The disclosure also provides a pharmaceutical preparation
comprising a subject chimeric protein and a pharmaceutically
acceptable carrier. A pharmaceutical preparation may be employed to
promote growth of a tissue or diminishing or prevent loss of a
tissue in a subject, preferably a human. The targeted tissue can
be, for example, bone, cartilage, skeletal muscle, cardiac muscle
and/or neuronal tissue.
[0146] In another aspect, a chimeric TGF-beta polypeptide can be
formulated either alone or in combination with other agents for
administration (e.g., as a lotion, cream, spray, gel, or ointment).
It may be formulated into liposomes to reduce toxicity or increase
bioavailability. Other methods for delivery include oral methods
that entail encapsulation of the in microspheres or proteinoids,
aerosol delivery (e.g., to the lungs), or transdermal delivery
(e.g., by iontophoresis or transdermal electroporation). Other
methods of administration will be known to those skilled in the
art.
[0147] Preparations for parenteral administration of a composition
comprising a chimeric TGF-beta polypeptide include sterile aqueous
or non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils (e.g., olive oil), and injectable organic esters
such as ethyl oleate. Examples of aqueous carriers include water,
saline, and buffered media, alcoholic/aqueous solutions, and
emulsions or suspensions. Examples of parenteral vehicles include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride, lactated Ringer's, and fixed oils. Intravenous vehicles
include fluid and nutrient replenishers, electrolyte replenishers
(such as those based on Ringer's dextrose), and the like.
Preservatives and other additives such as, other antimicrobial,
anti-oxidants, cheating agents, inert gases and the like also can
be included.
[0148] The disclosure provides various disease and disorders that
may be modulated by a TGF-beta protein family member comprising
contacting or administering a therapeutically effective amount of a
chimeric TGF-beta polypeptide either alone or in combination with
other agents to a subject who has, or is at risk of having, such a
disorder.
[0149] A therapeutically effective amount can be measured as the
amount sufficient to decrease a subject's symptoms associated with
the diseases or disorder. Typically, the subject is treated with an
amount of a therapeutic composition sufficient to reduce a symptom
of a disease or disorder by at least 50%, 90% or 100%. Generally,
the optimal dosage will depend upon the disorder and factors such
as the weight of the subject, the age, the weight, sex, and degree
of symptoms. For example, with respect to bone morphogenesis,
optionally, the dosage may vary with the type of matrix used in the
reconstitution and the types of compounds in the composition. The
addition of other known growth factors to the final composition,
may also affect the dosage. Progress can be monitored by periodic
assessment of bone growth and/or repair, for example, X-rays,
histomorphometric determinations, and tetracycline labeling.
Nonetheless, suitable dosages can readily be determined by one
skilled in the art. Typically, a suitable dosage is 0.5 to 40 mg/kg
body weight, e.g., 1 to 8 mg/kg body weight.
[0150] As mentioned previously, the compositions and methods of the
disclosure can include the use of additional (e.g., in addition to
a chimeric TGF-beta polypeptide) therapeutic agents (e.g., an
inhibitor of TNF, an antibiotic, and the like). The chimeric
TGF-beta polypeptide, other therapeutic agent(s), and/or
antibiotic(s) can be administered, simultaneously, but may also be
administered sequentially.
[0151] A pharmaceutical composition comprising a chimera according
to the disclosure can be in a form suitable for administration to a
subject using carriers, excipients, and additives or auxiliaries.
Frequently used carriers or auxiliaries include magnesium
carbonate, titanium dioxide, lactose, mannitol and other sugars,
talc, milk protein, gelatin, starch, vitamins, cellulose and its
derivatives, animal and vegetable oils, polyethylene glycols and
solvents, such as sterile water, alcohols, glycerol, and polyhydric
alcohols. Intravenous vehicles include fluid and nutrient
replenishers. Preservatives include antimicrobial, anti-oxidants,
chelating agents, and inert gases. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the like,
as described, for instance, in Remington's Pharmaceutical Sciences,
15th ed., Easton: Mack Publishing Co., 1405-1412, 1461-1487 (1975),
and The National Formulary XIV., 14th ed., Washington: American
Pharmaceutical Association (1975), the contents of which are hereby
incorporated by reference. The pH and exact concentration of the
various components of the pharmaceutical composition are adjusted
according to routine skills in the art. See Goodman and Gilman's,
The Pharmacological Basis for Therapeutics (7th ed.).
[0152] The pharmaceutical compositions according to the disclosure
may be administered locally or systemically. A "therapeutically
effective dose" is the quantity of an agent according to the
disclosure necessary to prevent, to cure, or at least partially
arrest a symptoms associated with a disease or disorder or to
promote cell growth, proliferation or differentiation. Amounts
effective for this use will, of course, depend on the severity of
the disease, disorder, or desired effect and will depend on weight
and general state of the subject. Typically, dosages used in vitro
may provide useful guidance in the amounts useful for in situ
administration of the pharmaceutical composition, and animal models
may be used to determine effective dosages for treatment of
infections. Various considerations are described, e.g., in Langer,
Science, 249: 1527, (1990); Gilman et al. (eds.) (1990), each of
which is herein incorporated by reference. Dosages of
pharmaceutically active compounds can be determined by methods
known in the art, see, e.g., Remington's Pharmaceutical Sciences
(Maack Publishing Co., Easton, Pa.); Remington, The Science &
Practice of Pharmacy, (Lippincott Williams & Wilkins; Twenty
first Edition). The therapeutically effective dosage of any
specific compound will vary somewhat from compound to compound, and
patient to patient, and will depend upon the condition of the
patient and the route of delivery. As a general proposition, a
dosage from about 0.1 to about 100 mg/kg will have therapeutic
efficacy, with all weights being calculated based upon the weight
of the compound, including the cases where a salt is employed.
Toxicity concerns at the higher level can restrict intravenous
dosages to a lower level such as up to about 10 to about 20 mg/kg,
with all weights being calculated based upon the weight of the
compound, including the cases where a salt is employed. A dosage
from about 10 mg/kg to about 50 mg/kg can be employed for oral
administration. Typically, a dosage from about 0.5 mg/kg to 15
mg/kg can be employed for intramuscular injection. Particular
dosages are about 1 .mu.mol/kg to 50 .mu.mol/kg, and more
particularly to about 22 .mu.mol/kg and to 33 .mu.mol/kg of the
compound for intravenous or oral administration, respectively.
[0153] In particular embodiments of the disclosure, more than one
administration (e.g., two, three, four, or more administrations)
can be employed over a variety of time intervals (e.g., hourly,
daily, weekly, monthly, etc.) to achieve therapeutic effects.
[0154] The compositions and chimera of the disclosure find use in
veterinary and medical applications. Suitable subjects include both
avians and mammals, with mammals being preferred. The term "avian"
as used herein includes, but is not limited to, chickens, ducks,
geese, quail, turkeys, and pheasants. The term "mammal" as used
herein includes, but is not limited to, humans, bovines, ovines,
caprines, equines, felines, canines, lagomorphs, etc. Human
subjects include neonates, infants, juveniles, and adults. In other
embodiments, the subject is an animal model of bone disease.
[0155] As used herein, "administering a therapeutically effective
amount" is intended to include methods of giving or applying a
pharmaceutical composition of the disclosure to a subject that
allow the composition to perform its intended therapeutic
function.
[0156] The pharmaceutical composition can be administered in a
convenient manner, such as by injection (subcutaneous, intravenous,
etc.), oral administration, inhalation, transdermal application, or
rectal administration. Depending on the route of administration,
the pharmaceutical composition can be coated with a material to
protect the pharmaceutical composition from the action of enzymes,
acids, and other natural conditions that may inactivate the
pharmaceutical composition. The pharmaceutical composition can also
be administered parenterally or intraperitoneally. Dispersions can
also be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof, and in oils. Under ordinary conditions of storage
and use, these preparations may contain a preservative to prevent
the growth of microorganisms.
[0157] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. In all cases, the
composition should be sterile and should be fluid to the extent
that easy syringability exists. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol, polyol
(for example, glycerol, propylene glycol, and liquid polyetheylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size, in the case of dispersion, and by the use
of surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be typical to
include isotonic agents, for example, sugars, polyalcohols, such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent that delays
absorption, for example, aluminum monostearate and gelatin.
[0158] Sterile injectable solutions can be prepared by
incorporating the pharmaceutical composition in the required amount
in an appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the
pharmaceutical composition into a sterile vehicle that contains a
basic dispersion medium and the required other ingredients from
those enumerated above.
[0159] The pharmaceutical composition can be orally administered,
for example, with an inert diluent or an assimilable edible
carrier. The pharmaceutical composition and other ingredients can
also be enclosed in a hard or soft-shell gelatin capsule,
compressed into tablets, or incorporated directly into the
individual's diet. For oral therapeutic administration, the
pharmaceutical composition can be incorporated with excipients and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations should contain at least 1% by weight
of active compound. The percentage of the compositions and
preparations can, of course, be varied and can conveniently be
between about 5% to about 80% of the weight of the unit.
[0160] The tablets, troches, pills, capsules, and the like can also
contain the following: a binder, such as gum gragacanth, acacia,
corn starch, or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent, such as corn starch, potato starch, alginic
acid, and the like; a lubricant, such as magnesium stearate; and a
sweetening agent, such as sucrose, lactose or saccharin, or a
flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring. When the dosage unit form is a capsule, it can contain,
in addition to materials of the above type, a liquid carrier.
Various other materials can be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules can be coated with shellac, sugar, or both. A
syrup or elixir can contain the agent, sucrose as a sweetening
agent, methyl and propylparabens as preservatives, a dye, and
flavoring, such as cherry or orange flavor. Of course, any material
used in preparing any dosage unit form should be pharmaceutically
pure and substantially non-toxic/biocompatible in the amounts
employed. In addition, the pharmaceutical composition can be
incorporated into sustained-release preparations and
formulations.
[0161] Thus, a "pharmaceutically acceptable carrier" is intended to
include solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like. The use of such media and agents for pharmaceutically active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the pharmaceutical
composition, use thereof in the therapeutic compositions and
methods of treatment is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
[0162] In certain embodiments, the therapeutic method of the
disclosure includes administering the composition topically,
systemically, or locally as an implant or device. When
administered, the therapeutic composition described by the
disclosure are generally in a pyrogen-free, physiologically
acceptable form. Further, the composition may desirably be
encapsulated or injected in a viscous form for delivery to the site
of bone, cartilage or tissue damage. Topical administration may be
suitable for wound healing and tissue repair. Therapeutically
useful agents other than the chimeras of the disclosure may also
optionally be included in the composition as described above, may
alternatively or additionally, be administered simultaneously or
sequentially with the chimeras in the methods of the described
herein. For example, preferably for bone and/or cartilage
formation, the composition would include a matrix capable of
delivering BMP chimeras or other therapeutic compounds to the site
of bone and/or cartilage damage, providing a structure for the
developing bone and cartilage and optimally capable of being
resorbed into the body. For example, the matrix may provide slow
release of the BMP chimeras. Such matrices may be formed of
materials presently in use for other implanted medical
applications.
[0163] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. The particular application of the subject
compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined calcium sulfate, tricalciumphosphate, hydroxyapatite,
polylactic acid and polyanhydrides. Other potential materials are
biodegradable and biologically well defined, such as bone or dermal
collagen. Further matrices are comprised of pure proteins or
extracellular matrix components. Other potential matrices are
non-biodegradable and chemically defined, such as sintered
hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices
may be comprised of combinations of any of the aforementioned types
of material, such as polylactic acid and hydroxyapatite or collagen
and tricalciumphosphate. The bioceramics may be altered in
composition, such as in calcium-aluminate-phosphate and processing
to alter pore size, particle size, particle shape, and
biodegradability.
[0164] Certain compositions disclosed herein may be administered
topically, either to skin or to mucosal membranes. The topical
formulations may further include one or more of the wide variety of
agents known to be effective as skin or stratum corneum penetration
enhancers. Examples of these are 2-pyrrolidone,
N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide,
propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide,
and azone. Additional agents may further be included to make the
formulation cosmetically acceptable. Examples of these are fats,
waxes, oils, dyes, fragrances, preservatives, stabilizers, and
surface active agents. Keratolytic agents such as those known in
the art may also be included. Examples are salicylic acid and
sulfur.
[0165] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. "Dosage unit form" as used herein, refers to
physically discrete units suited as unitary dosages for the
individual to be treated; each unit containing a predetermined
quantity of pharmaceutical composition is calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the disclosure are related to the characteristics of the
pharmaceutical composition and the particular therapeutic effect to
be achieve.
[0166] The principal pharmaceutical composition is compounded for
convenient and effective administration in effective amounts with a
suitable pharmaceutically acceptable carrier in an acceptable
dosage unit. In the case of compositions containing supplementary
active ingredients, the dosages are determined by reference to the
usual dose and manner of administration of the said
ingredients.
[0167] One of the challenges to using chimeras as therapeutics is
the ability to deliver the proteins effectively. The chimeras of
the disclosure can be delivered by several different methods. In
the blood stream, the half-life of most TGF-.beta. ligands is on
the order of minutes. To compensate for the ligands being degraded
so quickly, current therapies involving TGF-.beta. ligands use very
high doses of the proteins. Alternatively, several means to
directly modify the ligands or delivery systems are available to
help improve the stability or sustained release properties of the
ligands.
[0168] (1) Direct modification of the protein includes PEGylation
as one common form of modification. In this method, polyethylene
glycol (PEG) is covalently attached to the protein in hopes of
improving stability by increasing solubility, resistance to
proteolysis, and decreased immunogenicity.
[0169] (2) Rational modification of residues on the protein
surface. By improving any electrostatic instability, without
changing overall protein function, the overall stability of
molecule can be improved. Using continuum electrostatic models,
residues contributing to instability can be located and then
analyzed to see if it can be mutated to a more favorable
residue.
[0170] (3) Fusing the ligand to another protein or portion of a
protein is another technique to increase protein stability and
solubility. The antibody constant fragment (Fc) is common fusion
partner used to improve the stability and solubility.
[0171] (4) The use of liposomes can be used as a protein delivery
vehicle. Liposomes are composed of any number of different
phospholipids, which self assemble to form spheres. The protein of
interest is encapsulated inside the bilayer, protecting it from the
outside environment. The phospholipid composition influences the
exact properties of the liposome and can be tailored to release the
protein under any number of desired conditions. Polymer/liposome
composite systems are also available to be used as delivery
systems. Ideally, this type of system combines the advantages of
each system to improve protein delivery.
[0172] (5) Similar to liposomes, polymers can be used as protein
drug delivery systems. The polymers are used to make a matrix,
commonly what is termed a hydrogel due to the high water content of
the material. The advantage of using the gel is it allows for long
term, sustained release as well as protecting the protein from
proteolysis. As with the liposomes, the polymers used to make the
gel influence its properties. There are two general classifications
for the materials used to make the hydorgels: natural and unnatural
polymers. Common materials used to create hydrogels using natural
polymers include collagen, gelatin, fibrin, Hyaluronic acid,
alginate, chitosan, and dextran. Synthetic polymers used to make
hydrogels include Poly(ethylene oxide), Poly(acrylic acid),
Poly(N-isopropylacrylamide), Poly(vinyl alcohol), and
Polyphosphazene.
[0173] (6) A different kind of hydrogel can be created without the
use of polymers, either natural or unnatural. Considered to be a
bioactive glass, or Xerogel, this material is created from silica
and calcium phosphate layer capable of absorbing the protein of
interest. See, e.g., FIG. 8. The Xerogel increases the sustained
release time of the protein up to weeks. FIG. 1 shows results from
cell viability assay using osteoblast cell line MC3T3 by MTT assay,
which shows that the xerogel material is nontoxic up to the highest
concentration of 30 mg/ml in the culture media we tested.
[0174] Chimera of the disclosure alone or in combination with a
pharmaceutically acceptable carrier can be used to treat any number
of disease and disorder or modulate cellular or tissue
activity.
[0175] The chimeric polypeptides of the disclosure can be used to
treat any number of disease or disorders where modulating of
TGF-beta activity provides a therapeutic benefit. For example, the
chimera of the disclosure can be used in subjects suffering from
osteoporosis, cartilage disease or periodontal diseases. The
chimera can be used to promote bone and/or cartilage formation,
inhibiting bone loss/density or demineralization, promoting bone
deposition and the like. Alternatively, the chimera can be used to
inhibit excessive bone density and growth. In other embodiment, the
chimera can be used in the treatment of endocrine diseases and
disorders, hyperparathyroidism, Cushing's disease, malabsorption,
renal tubular acidosis, or thyrotoxicosis.
[0176] The chimera of the disclosure can also be used in the
treatment or modulating of sexual development, pituitary hormone
production, and creation of bone and cartilage. The chimera can
also be used for the treatment of cell proliferative diseases and
disorders, cell growth and differentiation associated with
inflammation, allergy, autoimmune diseases, infectious diseases,
and tumors.
[0177] In a further aspect, the chimera of the disclosure can be
used in the treatment of neuromuscular disorders, such as muscular
dystrophy and muscle atrophy, congestive obstructive pulmonary
disease, muscle wasting syndrome, obesity or other metabolic
diseases including, for example, type 2 diabetes.
[0178] The chimera of the disclosure can be used in degenerative
muscle diseases characterized by abnormal amount, development or
metabolic activity of muscle tissue, including gradual weakening
and deterioration of skeletal muscles. Examples of muscle disease
and disorders include, but are not limited to, a muscle wasting
disorder, cachexia, anorexia, AIDS wasting syndrome, muscular
dystrophies, Duchenne Muscular Dystrophy (DMD), Becker Muscular
Dystrophy (BMD), Myotonic Dystrophy (MMD) (also known as Steinert's
Disease), Oculopharyngeal Muscular Dystrophy (OPMD), Emery-Dreifuss
Muscular Dystrophy (EDMD), Limb-Girdle Muscular Dystrophy (LGMD),
Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) (also known as
Landouzy-Dejerine), Congenital Muscular Dystrophy (CMD), and Distal
Muscular Dystrophy (DD).
[0179] The chimera of the disclosure can be used in methods and
compositions to prevent, treat, or alleviate symptoms of a
neurodegenerative disease or disorder including, but not limited
to, Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic
Lateral Sclerosis (ALS), and Huntington's disease (HD), and other
neuromuscular diseases, motor neuron diseases, diseases of the
neuromuscular junction, and/or inflammatory myopathies.
[0180] A subject may have a disorder associated with abnormal cell
growth and differentiation which may cause inflammation, allergy,
autoimmune diseases, infectious diseases, and/or tumors. A subject
may have a heart disorder, such as a disorder associated with
excessive cardiomyocyte proliferation or growth, or a disorder in
which it would be desirable to stimulate cardiomyocyte growth or
proliferation. Subject chimeric TGF-beta superfamily proteins may
be designed for the treatment of essentially any disorder that is
amenable to treatment by agonists or antagonists of a member of the
TGF-beta superfamily.
[0181] The following examples are meant to further explain, but not
limited the foregoing disclosure or the appended claims.
EXAMPLES
Example 1
[0182] Generation of TGF-.beta. Chimeras. To generate these novel
TGF-.beta. ligands, a modified directed evolution approach was
utilized. Typically, this technique involves making a large number
of random protein sequences, greater than 10.sup.3, either by
mixing the sequences of homologous genes or inserting random
mutations and then screening for the desired ligand properties. In
one set of experiments, sequences that were known to refold
efficiently, termed the backbone ligand, were combined with a
second ligand sequence containing signaling properties desire to
mimic the target ligand. Using a structure guided approach, several
TGF-.beta. ligand crystal structures were analyzed and divided into
6 distinct sections. These sections roughly encompass the following
regions of the ligand: section 1, N-terminus and beta strand 1;
section 2, beta strand 2; section 3, pre-helix loop; section 4,
alpha helix; section 5, beta strand 3; and section 6, beta stand 4
and C-terminus. Using this protocol, 64 different ligand
combinations are possible for each set of TGF-.beta. ligands chosen
to be recombined. When two or more parental chains are from
different subfamilies (e.g. BMP/GDF v.s. TGFbeta), the difference
between their signaling mechanisms may not be captured if sections
3 and 4 are separated. To be broadly applicable as the design
principle, it is also part of the design to keep two structural
segments, sections 3 and 4, can be treated as one section of either
of the parental gene (referred to as section 3*4).
[0183] The strategy was implemented by making activin/BMP-2
chimeras using activin-.beta.A as a target ligand and BMP-2 as the
backbone ligand. Activin-.beta.A was picked as the target ligand as
it is biologically very interesting. BMP-2 was chosen as the
backbone ligand because it has been shown to refold with excellent
efficiency, >10% dimer yield from starting denatured inclusion
bodies, and these dimers have been shown to be active in both in
vitro and in vivo experiments. To design the various sections, a
sequence alignment of BMP-2 and activin-.beta.A was performed to
locate regions of sequence identity between the ligands (FIG. 7).
These regions were used as the boundaries for the different
sections. By using these parts of the sequence as the overlap
regions for the oligonucleotides during PCR changes will not be
introduced into either the BMP-2 or activin-.beta.A sequences. The
sequence alignment was then used in conjunction with data from
previously solved BMP-2 and activin-.beta.A structures to
ultimately determine the 6 sections (FIGS. 7a-c). Due to
limitations with regions of identity between the sequences, the
sections had to be shifted slightly from ideal. Particularly, the
pre-helix loop and the majority of the a-helix were combined into
one section, while the remainder of the a-helix to the beginning of
beta strand 3 was placed into a different section (FIGS. 7b and c).
Additionally, 3-point mutations were inserted to allow for the
cloning strategy to be successful. At the end of section 3, the
BMP-2 sequence is TLVN, while the activin-.beta.A sequence is TVIN
(FIG. 7a). Since these residue differences are conservative, the
leucine and valine from the BMP-2 were introduced into the
corresponding activin-.beta.A sequence. The third mutation is found
at the end of section 5. Here, the BMP-2 sequence is LYLD, while
the equivalent activin-.beta.A sequence is LYYD (FIG. 7a). Since
this residue difference is less conserved than the previous two,
the tyrosine from activin-.beta.A was inserted into the
corresponding BMP-2 sequence.
[0184] The N-terminus of activin-.beta.A contains 2 additional
cysteines (FIG. 7a) which form a 4.sup.th intra-disulfide bond. To
eliminate the potential of this extra disulfide bond complicating
the refolding process, the section which contained these residues
was eliminated from section 1 of activin-.beta.A chimera
design.
[0185] For the activin/BMP-2 chimeras, the mature domains of human
BMP-2 and human activin-.beta.A were initially divided into 6
sections each and primers were designed for each section. For
BMP-2, the primers coded for the following protein sequences:
Section 1, QAKHKQRKRLKSSCKRHPLYVDFSDVGWND (SEQ ID NO: 98); Section
2, WIVAPPGYHAFYCHGECP (SEQ ID NO: 99); Section 3,
FPLADHLNSTNHAIVQTLVN (SEQ ID NO: 100); Section 4, SVNSKIPKACCVP
(SEQ ID NO: 101); Section 5, TELSAISMLYYD (SEQ ID NO: 102); Section
6, ENEKVVLKNYQDMVVEGCGCR (SEQ ID NO: 103). For activin-.beta.A, the
primers coded for the following protein sequences: Section 1,
RGLECDGKVNICCKKQFFVSFKDIGWNDW (SEQ ID NO: 104); Section 2,
WIIAPSGYHANYCEGECP (SEQ ID NO: 105); Section 3, SHIAGTSGSSLSFHSTLVN
(SEQ ID NO: 106); Section 4, HYRMRGHSPFANLKSCCVP (SEQ ID NO: 107);
Section 5, TKLRPMSMLYYD (SEQ ID NO: 108); Section 6,
DGQNIIKKDIQNMIVEECGCS (SEQ ID NO: 109). An overlapping PCR strategy
was used to mix the various sections together to generate full
length chimeras. To generate the 1b chimeras, two oligos were used
to insert the BMP-2 sequence QAKHKQRKRLKSSCKRHPLYVDFSDVGWNDII (SEQ
ID NO: 110) into the target gene. Outer primers for all constructs
were constructed to incorporate a 5' NdeI site and a 3' XhoI site
for cloning into pET21a expression vector. The desired protein
sequences were confirmed by DNA sequencing.
[0186] The chimeras were labeled according to the sections they
contained. For example, 1b2b3b4a5a6b, in which the b's represent
that the section was taken from BMP-2 and the a's represent that
the section was derived from activin-.beta.A. The chimeras were
also given shorthand numeric designations, such as A/B2-020, so
that any functional assays could be undertaken in a blind manner.
Table 1 sets forth some of the various chimeras:
TABLE-US-00003 TABLE 1 BMP-2/activin Sample Exemplary constructs
Designation DNA Sequence Protein Sequence Characteristics
1b2b3a4a5a6a AB2-001 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H
K Q R K R L K S S C K R H P L Potential universal antagonist
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I V A
P P G Y H because it competes AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC
A F Y C H G E C P S H I A G T S G S S L receptor binding
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC S F H S T L V N H Y R M R G H
S P F A N but not signaling. CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT
L K S C C V P T K L R P M S M L Y Y D D Acts as neither
CCTTCCACTCAACGTTGGTCAACCACTACCGCATGCG G Q N I I K K D I Q N M I V E
E C G C S BMP-2 nor activin-.beta.A.
GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT (SEQ ID NO: 13)
GTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACT
ATGATGATGGTCAAAACATCATCAAAAAGGACATTC
AGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 12) 1b2b3a4a5b6a
AB2-002 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L Activity in stem cell
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I V A
P P G Y H differentiation AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F
Y C H G E C P S H I A G T S G S S L assays unlike BMP-2.
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC S L H S T L V N H Y R M R G H
S P F A N CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT L K S C C V P T E L
S A I S M L Y Y D D CCTTACACTCAACGTTGGTCAACCACTACCGCATGCG G Q N I I
K K D I Q N M I V E E C G C S GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT
(SEQ ID NO: 15): GTCCCGACAGAGCTCAGTGCTATCTCGATGTTGTACT
ATGATGATGGTCAAAACATCATCAAAAAGGACATTC
AGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 14) 1b2a3a4a5b6a
AB2-003 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V
G W N D W I I A P S G Y H AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N
Y C E G E C P S H I A G T S G S S L
TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC S F H S T L V N H Y R M R G H
S P F A N CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT L K S C C V P T E L
S A I S M L Y Y D D CCTTCCACTCAACGTTGGTCAACCACTACCGCATGCG G Q N I I
K K D I Q N M I V E E C G C S GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT
(SEQ ID NO: 27) GTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACT
ATGATGATGGTCAAAACATCATCAAAAAGGACATTC
AGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 26) 1b2a3b4b5a6a
AB2-004 ATGCAAGCCAAACACAAACAGCGGAAGCGTCTTAAG M Q A K H K Q R K R L
K S S C K R H P L `Super" BMP-2 activity,
TCCAGCTGCAAAAGGCACCCTTTGTATGTGGACTTCA Y V D F S D V G W N D W I I A
P S G Y H unable to be GTGATGTGGGGTGGAATGACTGGATCATTGCTCCCT A N Y C
D G E C P F P L A D H L N S T N inhibited by Noggin
CTGGCTATCATGCCAACTACTGCGACGGAGAATGCC H A I V Q T L V N S V N S K I
P K A C C CTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCA V P T K L R P M S M
L Y Y D D G Q N I I TGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCT K K D I Q
N M I V E E C G C S AAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTG (SEQ ID
NO: 31) AGACCCATGTCCATGTTGTACTATGATGATGGTCAA
AACATCATCAAAAAGGACATTCAGAACATGATCGTG GAGGAGTGTGGGTGCTCA (SEQ ID NO:
30) 1b2b3b4b5b6b AB2-005 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L AB2-005 (BMP-2.sub.mq)
(BMP-2.sub.mq) GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V
G W N D W I V A P P G Y H contains one amino
AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F Y C H G E C P F P L A D H
L N S T N acid (Met) added CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC H A
I V Q T L V N S V N S K I P K A C C at the N-terminus
CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC V P T E L S A I S M L Y L D E
N E K V V of mature BMP-2 in ATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTC
L K N Y Q D M V V E G C G C R nature. Met originates
TAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACT (SEQ ID NO: 14) from the
translation CAGTGCTATCTCGATGCTGTACCTTGACGAGAATGA initiation codon
(ATG). AAAGGTTGTATTAAAGAACTATCAGGACATGGTTGT Unless it is truncated
GGAGGGTTGTGGGTGTCGC (SEQ ID NO: 1) during the folding pro- cess, it
can remain as the N-terminus of AB2- 005. Activity in stem cell
differentiation assays unlike BMP-2. 1b2a3a4a5b6b AB2-006
ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K
R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D
W I I A P S G Y H AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C E G
E C P S H I A G T S G S S L TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC S
F H S T L V N H Y R M R G H S P F A N
CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT L K S C C V P T E L S A I S M
L Y L D E CCTTCCACTCAACGTTGGTCAACCACTACCGCATGCG N E K V V L K N Y Q
D M V V E G C G C R GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT (SEQ ID
NO: 25) GTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACC
TTGACGAGAATGAAAAGGTTGTATTAAAGAACTATC
AGGACATGGTTGTGGAGGGTTGTGGGTGTCGC (SEQ ID NO: 24) 1b2a3a4a5a6b
AB2-007 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L Activity in stem cell
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H differentiation AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N
Y C E G E C P S H I A G T S G S S L assays unlike BMP-2.
TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC S F H S T L V N H Y R M R G H
S P F A N CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT L K S C C V P T K L
R P M S M L Y L D E CCTTCCACTCAACGTTGGTCAACCACTACCGCATGCG N E K V V
L K N Y Q D M V V E G C G C R GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT
(SEQ ID NO: 23) GTCCCGACCAAGCTGAGACCCATGTCCATGTTGTAC
CTTGACGAGAATGAAAAGGTTGTATTAAAGAACTAT
CAGGACATGGTTGTGGAGGGTTGTGGGTGTCGC (SEQ ID NO: 22) 1b2a3a4a5a6a
AB2-008 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L Functions like
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H activin-.beta.A in cell
AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C E G E C P S H I A G T
S G S S L signaling and in vivo
TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC S F H S T L V N H Y R M R G H
S P F A N experiments, ~4-fold
CCTTCCCATATAGCAGGCACGTCCGGGTCCTCACTGT L K S C C V P T K L R P M S M
L Y Y D D lower potency; replaces
CCTTCCATTCAACGTTGGTCAACCACTACCGCATGCG G Q N I I K K D I Q N M I V E
E C G C S TGF-beta 1 in chemically-
GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT (SEQ ID NO: 17) defined stem
cell GTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACT media containing FGF2.
ATGATGATGGTCAAAACATCATCAAAAAGGACATTC
AGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 16) 1b2a3a4a5a6a
AB2-009 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L Functions like activin-.beta.A L66V/V67I
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H in cell signaling and
AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C E G E C P S H I A G T
S G S S L in vivo experiments, TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC
S F H S T V I N H Y R M R G H S P F A N ~10-fold lower A
CCTTCCCATATAGCAGGCACGTCCGGGTCCTCACTGT L K S C C V P T K L R P M S M
L Y Y D D potency in activin-.beta.
CCTTCCATTCAACGGTGATCAACCACTACCGCATGCG G Q N I I K K D I Q N M I V E
E C G C S signaling activity. GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT
(SEQ ID NO: 19) GTCCCGACCAAGCTGAGACCCATGTCCATGTTGTACT
ATGATGATGGTCAAAACATCATCAAAAAGGACATTC
AGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 18) 1b(1a_II) AB2-010
ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K
K Q F F Functions like activin-.beta.A 2a3a4a5a6a
GTCCAGCTGTAAGAAACAGTTCTTTGTCAGTTTCAAG V S F K D I G W N D W I I A P
S G Y H A in cell signaling and
GACATCGGGTGGAATGACTGGATCATTGCTCCCTCT N Y C E G E C P S H I A G T S
G S S L S in vivo experiments, GGCTATCATGCCAACTACTGCGAGGGAGAATGCCCT
F H S T L V N H Y R M R G H S P F A N L ~20-fold lower potency in
TCCCATATAGCAGGCACGTCCGGGTCCTCACTGTCCT K S C C V P T K L R P M S M L
Y Y D D G activin-.beta.A signaling.
TCCATTCAACGTTGGTCAACCACTACCGCATGCGGG Q N I I K K D I Q N M I V E E
C G C S GCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGTGT (SEQ ID NO: 21)
CCCGACCAAGCTGAGACCCATGTCCATGTTGTACTAT
GATGATGGTCAAAACATCATCAAAAAGGACATTCAG AACATGATCGTGGAGGAGTGTGGGTGCTCA
(SEQ ID NO: 20) 1b2b3b4b5a6a AB2-011
ATGCAAGCCAAACACCAACAGCGGAAACGCCTTAAG M Q A K H Q Q R K R L K S S C
K R H P L `Super" BMP-2 activity,
TCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTCA Y V D F S D V G W N D W I V A
P P G Y H unable to be GTGACGTGGGGTGGAATGACTGGATTGTGGCTCCCC A F Y C
H G E C P F P L A D H L N S T N inhibited by Noggin
CGGGGTATCACGCCTTTTACTGCCACGGAGAATGCC H A I V Q T L V N S V N S K I
P K A C C CTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCA V P T K L R P S M L
Y Y D D G Q N I I K TGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCT K D I Q N
M I V E E C G C S AAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCTG (SEQ ID NO:
9) AGACCCTCCATGTTGTACTATGATGATGGTCAAAAC
ATCATCAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA (SEQ ID NO: 8)
1b2b3b4b5b6a AB2-012 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H
K Q R K R L K S S C K R H P L `Super" BMP-2 activity,
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I V A
P P G Y H partially AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F Y C H
G E C P F P L A D H L N S T N inhibited by Noggin
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC H A I V Q T L V N S V N S K I
P K A C C CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC V P T E L S A I S M
L Y Y D D G R N I I ATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTC K K D I Q
N M I V E E C G C S TAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACT (SEQ ID
NO: 7) CAGTGCTATCTCGATGTTGTACTATGATGATGGTCGA
AACATCATCAAAAAGGACATTCAGAACATGATCGTG GAGGAGTGTGGGTGCTCA (SEQ ID NO:
6) 1b2b3b4b5a6b AB2-013 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K
H K Q R K R L K S S C K R H P L Activity comparable to BMP-2,
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I V A
P P G Y H inhibited by Noggin AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC
A F Y C H G E C P F P L A D H L N S T N
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC H A I V Q T L V N S V N S K I
P K A C C CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC V P T K L R P M S M
L Y Y D E N E K V V ATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTC L K N Y Q
D M V V E G C G C R TAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCT (SEQ ID
NO: 11) GAGACCCATGTCCATGTTGTACTATGATGAGAATGA
AAAGGTTGTATTAAAGAACTATCAGGACATGGTTGT GGAGGGTTGTGGGTGTCGC (SEQ ID
NO: 10) 1b2a3b4b5a6b AB2-014 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M
Q A K H K Q R K R L K S S C K R H P L Activity comparable to BMP-2,
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H partially blocked by Noggin
AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C D G E C P F P L A D H
L N S T N TCTGGCTATCATGCCAACTACTGCGACGGAGAATGC H A I V Q T L V N S
V N S K I P K A C C CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC V P T K L
R P M S M L Y L D E N E K V V ATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTC
L K N Y Q D M V V E G C G C R TAAGATTCCTAAGGCATGCTGTGTCCCGACCAAGCT
(SEQ ID NO: 33) GAGACCCATGTCCATGTTGTACCTTGACGAGAATGA
AAAGGTTGTATTAAAGAACTATCAGGACATGGTTGT
GGAGGGTTGTGGGTGTCGC (SEQ ID NO: 32) 1b2a3b4b5b6a AB2-015
ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K
R H P L `Super" BMP-2 activity,
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H unable to be AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C
D G E C P F P L A D H L N S T N inhibited by Noggin
TCTGGCTATCATGCCAACTACTGCGACGGAGAATGC H A I V Q T L V N S V N S K I
P K A C C CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC V P T E L S A I S M
L Y Y D D G Q N I I ATGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTC K K D I Q
N M I V E E C G C S TAAGATTCCTAAGGCATGCTGTGTCCCGACAGAACT (SEQ ID
NO: 29) CAGTGCTATCTCGATGTTGTACTATGATGATGGTCAA
AACATCATCAAAAAGGACATTCAGAACATGATCGTG GAGGAGTGTGGGTGCTCA (SEQ ID NO:
28) 1b, AB2-016 ATGCAAGCCAAACACAAACAGCGGAAGCGTCTTAAG M Q A K H K Q
R K R L K S S C K R H P L Activity comparable to BMP-2, 2a3b4b5b6b
TCCAGCTGCAAAAGGCACCCTTTGTATGTGGACTTCA Y V D F S D V G W N D W I I A
P S G Y H inhibited by Noggin GTGATGTGGGGTGGAATGACTGGATCATTGCTCCCT
A N Y C E G E C P F P L A D H L N S T N
CTGGCTATCATGCCAACTACTGCGAGGGAGAATGCC H A I V Q T L V N S V N S K I
P K A C C CTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCA V P T E L S A I S M
L Y L D E N E K V V CGCCATTGTTCAGACGTTGGTCAACTCTGTTAACTCT L K N Y Q
D M V V E G C G C R AAGATTCCTAAGGCATGCTGTGTCCCGACAGAACTC (SEQ ID
NO: 112) AGTGCTATCTCGATGCTGTACCTTGACGAGAATGAA
AAGGTTGTATTAAAGAACTATCAGGACATGGTTGTG GAGGGTTGCGGGTGTCGT (SEQ ID NO:
111) 1b2b3b4a5a6a AB2-017 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I V A
P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F Y C H G E C P F
P L A D H L N S T N CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC H A I V Q
T L V N H Y R M R G H S P F A CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC
N L K S C C V P T K L R P M S M L Y Y D
ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCAT D G Q N I I K K D I Q N M I V
E E C G C GCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTG S
CTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTG (SEQ ID NO: 114)
TACTATGATGATGGTCAAAACATCATCAAAAAGGAC
ATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 113) 1b2b3b4a5b6b
AB2-018 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V
G W N D W I V A P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F
Y C H G E C P F P L A D H L N S T N
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC H A I V Q T L V N H Y R M R G
H S P F A CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC N L K S C C V P T E
L S A I S I L Y L D ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCAT E N E K V
V L K N Y Q D M V V E G C G C GCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTG
R CTGTGTCCCGACAGAACTCAGTGCTATCTCGATACTG (SEQ ID NO: 116)
TACCTTGACGAGAATGAAAAGGTTGTATTAAAGAAC
TATCAGGACATGGTTGTGGAGGGTTGTGGGTGTCGC (SEQ ID NO: 115) 1b2b3b4a5a6b
AB2-019 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V
G W N D W I V A P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F
Y C H G E C P F P L A D H L N S T N
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC H A I V Q T L V N H Y R M R G
H S P F A CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC N L K S C C V P T K
L R P M S M L Y Y D ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCAT E N E K V
V L K N Y Q D M V V E G C G C GCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTG
R CTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTG (SEQ ID NO: 118)
TACTATGATGAGAATGAAAAGGTTGTATTAAAGAAC
TATCAGGACATGGTTGTGGAGGGTTGCGGGTGTCGT (SEQ ID NO: 117) 1b2b3b4a5b6a
AB2-020 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V
G W N D W I V A P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F
Y C H G E C P F P L A D H L N S T N
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC H A I V Q T L V N H Y R M R G
H S P F A CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC N L K S C C V P T E
L S A I S M L Y Y D ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCAT D G Q N I
I K K D I Q N M I V E E C G C GCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTG
S CTGTGTCCCGACAGAACTCAGTGCTATCTCGATGTTG (SEQ ID NO: 120)
TACTATGATGATGGTCAAAACATCATCAAAAAGGAC
ATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 119) 1b2b3a4a5a6b
AB2-021 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L GTCCAGCTGTAAGAGACACCCTTTGTATGTGGACTTC Y V D F S D V
G W N D W I V A P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F
Y C H G E C P S H I A G T S G S S L
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC S F H S T L V N H Y R M R G H
S P F A N CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT L K S C C V P T K L
R P M S M L Y L D E CCTTCCACTCAACGTTGGTCAACCACTACCGCATGCG N E K V V
L K N Y Q D M V V E G C G C R GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT
(SEQ ID NO: 122) GTCCCGACCAAGCTGAGACCCATGTCCATGTTGTAC
CTTGACGAGAATGAAAAGGTTGTATTAAAGAACTAT
CAGGACATGGTTGTGGAGGGTTGTGGGTGTCGC (SEQ ID NO: 121) 1b2b3a4a5b6b
AB2-022 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V
G W N D W I V A P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F
Y C H G E C P S H I A G T S G S S L
CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC S F H S T L V N H Y R M R G H
S P F A N CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT L K S C C V P T E L
S A I S M L Y L D E CCTTCCACTCAACGTTGGTCAACCACTACCGCATGCG N E K V V
L K N Y Q D M V V E G C G C R GGGCCATAGCCCCTTTGCCAACCTCAAATCGTGCTGT
(SEQ ID NO: 124) GTCCCGACAGAACTCAGTGCTATCTCGATGTTGTACT
ATGATGAGAATGAAAAGGTTGTATTAAAGAACTATC AGGACATGGTTGTGGAGGGTTGC (SEQ
ID NO: 123) 1b2b3a4b5b6b AB2-023
ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K
R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D
W I V A P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F Y C H G
E C P S H I A G T S G S S L CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC S
F H S T L V N S V N S K I P K A C C V
CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT P T E L S A I S M L Y L D E N
E K V V L CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAA K N Y Q D M V V E G
C G C R GATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAG (SEQ ID NO: 126)
TGCTATCTCGATGCTGTACCTTGACGAGAATGAAAA
GGTTGTATTAAAGAACTATCAGGACATGGTTGTGGA GGGTTGCGGGTGTCGT (SEQ ID NO:
125) 1b2b3a4b5b6a AB2-024 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I V A
P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F Y C H G E C P S
H I A G T S G S S L CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC S F H S T
L V N S V N S K I P K A C C V CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT
P T E L S A I S M L Y Y D D G Q N I I K
CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAA K D I Q N M I V E E C G C S
GATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAG (SEQ ID NO: 128)
TGCTATCTCGATGTTGTACTATGATGATGGTCAAAAC
ATCATCAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA (SEQ ID NO:
127) 1b2b3a4b5a6a AB2-025 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I V A
P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F Y C H G E C P S
H I A G T S G S S L CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC S F H S T
L V N S V N S K I P K A C C V CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT
P T K L R P M S M L Y Y D D G Q N I I K
CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAA K D I Q N M I V E E C G C S
GATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAG (SEQ ID NO: 130)
ACCCATGTCCATGTTGTACTATGATGATGGTCAAAAC
ATCATCAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA (SEQ ID NO:
129) 1b2b3a4b5a6b AB2-026 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I V A
P P G Y H AGTGACGTGGGGTGGAATGACTGGATTGTGGCTCCC A F Y C H G E C P S
H I A G T S G S S L CCGGGGTATCACGCCTTTTACTGCCACGGAGAATGC S F H S T
L V N S V N S K I P K A C C V CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT
P T K L R P M S M L Y Y D E N E K V V L
CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAA K N Y Q D M V V E G C G C R
GATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAG (SEQ ID NO: 132)
ACCCATGTCCATGTTGTACTATGATGAGAATGAAAA
GGTTGTATTAAAGAACTATCAGGACATGGTTGTGGA GGGTTGCGGGTGTCGT (SEQ ID NO:
131) 1b2a3a4b5b6b AB2-027 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C E G E C P S
H I A G T S G S S L TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC S F H S T
L V N S V N S K I P K A C C V CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT
P T E L S A I S M L Y L D E N E K V V L
CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAA K N Y Q D M V V E G C G C R
GATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAG (SEQ ID NO: 134)
TGCTATCTCGATGTTGTACCTTGACGAGAATGAAAA
GGTTGTATTAAAGAACTATCAGGACATGGTTGTGGA GGGTTGTGGGTGTCGC (SEQ ID NO:
133) 1b2a3a4b5b6a AB2-028 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C E G E C P S
H I A G T S G S S L TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC S F H S T
L V N S V N S K I P K A C C V CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT
P T E L N A I S M L Y Y D D G Q N I I K
CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAA K D I Q N M I V E E C G C S
GATTCCTAAGGCATGCTGTGTCCCGACAGAACTCAA (SEQ ID NO: 136)
TGCTATCTCGATGTTGTACTATGATGATGGTCAAAAC
ATCATTAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA (SEQ ID NO:
135) 1b2a3a4b5a6a AB2-030 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C E G E C P S
H I A G T S G S S L TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC S F H S T
L V N S V N S K I P K A C C V CCTTCTCATATAGCAGGCACGTCCGGGTCCTCACTGT
P T K L R P M S M L Y Y D D G Q N I I K
CCTTCCACTCAACGTTGGTCAACTCTGTTAACTCTAA K D I Q N M I V E E C G C S
GATTCCTAAGGCATGCTGTGTCCCGACCAAGCTGAG (SEQ ID NO: 138)
ACCCATGTCCATGTTGTACTATGATGATGGTCAAAAC
ATCATCAAAAAGGACATTCAGAACATGATCGTGGAG GAGTGTGGGTGCTCA (SEQ ID NO:
137) 1b2a3b4a5a6a AB2-031 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P S G Y H AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N Y C E G E C P F
P L A D H L N S T N TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC H A I V Q
T L V N H Y R M R G H S P F A CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC
N L K S C C V P T K L R P M S M L Y Y D
ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCAT D G Q N I I K K D I Q N M I V
E E C G C GCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTG S
CTGTGTCCCGACCAAGCTGAGACCCATGTCCATGTTG (SEQ ID NO: 140)
TACTATGATGATGGTCAAAACATCATCAAAAAGGAC
ATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 139) 1b2a3b4a5b6a
AB2-032 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V
G W N D W I I A P S G Y H AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N
Y C E G E C P F P L A D H L N S T N
TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC H A I V Q T L V N H Y R M R G
H S P F A CCTTTTCCTCTGGCTGATCATCTGAACTCCACTAATC N L K S C C V P T E
L S A I S M L Y Y D ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCAT D G Q N I
I K K D I Q N M I V E E C G C GCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTG
S CTGTGTCCCGACAGAACTCAGTGCTATCTCGATGCTG (SEQ ID NO: 142)
TACCTTGACGATGGTCAAAACATCATCAAAAAGGAC
ATTCAGAACATGATCGTGGAGGAGTGTGGGTGCTCA (SEQ ID NO: 141) 1b2a3b4a5b6b
AB2-033 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K
S S C K R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V
G W N D W I I A P S G Y H AGTGACGTGGGGTGGAATGACTGGATCATTGCTCCC A N
Y C E G E C P F P L A D H L N S T N
TCTGGCTATCATGCCAACTACTGCGAGGGAGAATGC H A I V Q T L V N H Y R M R G
H S P F A CCTTTTCCTCTGGCTGATCATCTGAACTCTACTAATC N L K S C C V P T E
L S A I S M L Y L D ATGCCATTGTTCAGACGTTGGTCAACCACTACCGCAT E N E K V
V L K N Y Q D M V V E G C G C GCGGGGCCATAGCCCCTTTGCCAACCTCAAATCGTG
R CTGTGTCCCGACAGAACTCAGTGCTATCTCGATGCTG (SEQ ID NO: 144)
TACCTTGACGAGAATGAAAAGGTTGTATTAAAGAAC
TATCAGGACATGGTTGTGGAGGGTTGCGGGTGTCGT (SEQ ID NO: 143) 1b2a3b4a5a6b
AB2-034 ATGCAAGCCAAACACAAACAGCGGAAGCGTCTTAAG M Q A K H K Q R K R L
K S S C K R H P L TCCAGCTGCAAAAGGCACCCTTTGTATGTGGACTTCA Y V D F S D
V G W N D W I I A P S G Y H GTGATGTGGGGTGGAATGACTGGATCATTGCTCCCT A
N Y C D G E C P F P L A D H L N S T N
CTGGCTATCATGCCAACTACTGCGACGGAGAATGCC H A I V Q T L V N H Y R M R G
H S P F A CTTTTCCTCTGGCTGATCATCTGAACTCCACTAATCA N L K S C C V P T K
L R P M S M L Y Y D TGCCATTGTTCAGACGTTGGTCAACCACTACCGCATG E N E K V
V L K N Y Q D M V V E G C G C CGGGGCCATAGCCCCTTTGCCAACCTCAAATCATGCT
R GTGTCCCGACCAAGCTGAGACCCATGTCCATGTTGT (SEQ ID NO: 146)
ACTATGATGAGAATGAAAAGGTTGTATTAAAGAACT
ATCAGGACATGGTTGTGGAGGGTTGCGGGTGTCGT (SEQ ID NO: 145) BMP-2.sub.ma
BMP-2.sub.ma ATGGCTCAAGCCAAACACAAACAGCGGAAACGCCTT M A Q A K H K Q R
K R L K S S C K R H P BMP-2.sub.ma contains two
AAGTCCAGCTGTAAGAGACACCCTTTGTACGTGGAC L Y V D F S D V G W N D W I V
A P P G Y additional amino TTCAGTGACGTGGGGTGGAATGACTGGATTGTGGCT H A
F Y C H G E C P F P L A D H L N S T acids (Met-Ala) at the
CCCCCGGGGTATCACGCCTTTTACTGCCACGGAGAA N H A I V Q T L V N S V N S K
I P K A C N-terminal side of TGCCCTTTTCCTCTGGCTGATCATCTGAACTCCACTA
C V P T E L S A I S M L Y L D E N E K V mature BMP-2 in nature
ATCATGCCATTGTTCAGACGTTGGTCAACTCTGTTAA V L K N Y Q D M V V E G C G C
R (QAKH . . .). Met is CTCTAAGATTCCTAAGGCATGCTGTGTCCCGACAGA (SEQ ID
NO: 148) often truncated during
ACTCAGTGCTATCTCGATGCTGTACCTTGACGAGAAT the folding process,
GAAAAGGTTGTATTAAAGAACTATCAGGACATGGTT but Ala remains as the
GTGGAGGGTTGTGGGTGTCGC N-terminus of the final (SEQ ID NO: 147)
product. Either form is regarded as BMP-2.sub.ma. 1b_BMP7 NB2-BMP7
ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A K H K Q R K R L K S S C K
R H P L GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D
W I I A P E G Y A AGTGACGTGGGGTGGAATGACTGGATTATCGCGCCT A Y Y C E G
E C A F P L N S Y M N A T N GAAGGCTACGCCGCCTACTACTGTGAGGGGGAGTGT H
A I V Q T L V H F I N P E T V P K P C
GCCTTCCCTCTGAACTCCTACATGAACGCCACCAACC C A P T Q L N A I S V L Y F D
D S S N V ACGCCATCGTGCAGACGCTGGTCCACTTCATCAACC I L K K Y R N M V V
R A C G C H CGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGC (SEQ ID NO: 35)
AGCTCAATGCCATCTCCGTCCTCTACTTCGATGACAG
CTCCAACGTCATCCTGAAGAAATACAGAAACATGGT GGTCCGGGCCTGTGGCTGCCAC (SEQ ID
NO: 34) 1b_BMP9 NB2-BMP9 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P K E Y E AGTGACGTGGGGTGGAATGACTGGATTATTGCCCCA A Y E C K G G C F F
P L A D D V T P T K AAAGAGTACGAGGCATACGAGTGTAAGGGCGGCTGT H A I V Q
T L V H L K F P T K V G K A C TTCTTTCCGCTGGCCGACGATGTCACCCCGACCAAGC
C V P T K L S P I S V L Y K D D M G V P
ACGCAATTGTCCAAACCTTAGTGCACCTGAAGTTCCC T L K Y H Y E G M S V A E C G
C R AACGAAAGTGGGTAAGGCATGTTGTGTGCCAACCAA (SEQ ID NO: 37)
GTTATCTCCAATTAGCGTGCTGTATAAGGATGATATG
GGCGTGCCGACGTTAAAGTATCATTACGAGGGCATG AGCGTCGCAGAGTGTGGCTGCCGC (SEQ
ID NO: 36) 1b_GDF7 NB2-GDF7 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q
A K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P L D Y E AGTGACGTGGGGTGGAATGACTGGATTATCGCGCCG A Y H C E G L C D F
P L R S H L E P T N CTGGACTACGAGGCGTACCACTGCGAGGGCCTATGC H A I I Q
T L V N S M A P D A A P A S C GATTTTCCTCTGCGTTCGCACCTCGAACCCACCAACC
C V P A R L S P I S I L Y Y D A A N N V
ATGCCATCATTCAGACGTTGGTCAACTCCATGGCACC V Y K Q Y E D M V V E A C G C
R AGACGCGGCGCCGGCCTCCTGCTGTGTCCCGGCGCG (SEQ ID NO: 39)
CCTCAGCCCCATCAGCATCTTGTACTATGATGCCGCC
AACAACGTTGTCTACAAGCAATACGAGGACATGGTG GTGGAGGCCTGTGGGTGTCGC (SEQ ID
NO: 38) 1b_G DF8 NB2-GDF8 ATGCAAGCCAAACACAAACAGCGGAAACGCCTTAA M Q A
K H K Q R K R L K S S C K R H P L
GTCCAGCTGTAAGAGACACCCTTTGTACGTGGACTTC Y V D F S D V G W N D W I I A
P K R Y K AGTGACGTGGGGTGGAATGACTGGATTATTGCACCC A N Y C S G E C E F
V F L Q K Y P H T H AAAAGATATAAGGCCAATTACTGCTCTGGAGAGTGT L V H Q A
N P R G S A G P C C T P T K M GAATTTGTATTTTTACAAAAATACCCTCACACTCATC
S P I N M L Y F N G K E Q I I Y G K I P
TTGTGCACCAAGCAAACCCCAGAGGTTCAGCAGGCC A M V V D R C G C S
CCTGCTGTACTCCCACAAAGATGTCTCCAATCAATAT (SEQ ID NO: 41)
GCTATATTTTAATGGCAAAGAACAAATAATATATGG
GAAAATTCCAGCCATGGTAGTAGATCGCTGTGGGTG CTCA (SEQ ID NO: 40) BMP2/BMP6
B2/B6 BMP-2.sub.wt and BMP-6 are BMP-2.sub.wt sequence is reported
Has increased SMAD- added together during above as AB2-005. BMP-6
amino mediated signaling the refolding to generate acid sequence.
Two amino acids, activity as compared the BMP2/BMP6 heterodimer.
MA, are present in BMP-2.sub.wt to either BMP-2 BMP-2.sub.wt
sequence herein is in contrast to mature form or BMP-6. also
reported as AB2-005. of BMP-2 existent in nature. BMP-6 DNA
sequence is: M Q Q S R N R S T Q S Q D V A R V S S A
ATGCAACAGAGTCGTAATCGCTCTACCCAGTCCCAG S D Y N S S E L K T A C R K H
E L Y V S GACGTGGCGCGGGTCTCCAGTGCTTCAGATTACAAC F Q D L G W Q D W I
I A P K G Y A A N Y AGCAGTGAATTGAAAACAGCCTGCAGGAAGCATGA C D G E C S
F P L N A H M N A T N H A I GCTGTATGTGAGTTTCCAAGACCTGGGATGGCAGGA V
Q T L V H L M N P E Y V P K P C C A P
CTGGATCATTGCACCCAAGGGCTATGCTGCCAATTA T K L N A I S V L Y F D D N S
N V I L K CTGTGATGGAGAATGCTCCTTCCCACTCAACGCACA K Y R N M V V R A C
G C H CATGAATGCAACCAACCACGCGATTGTGCAGACCTT (SEQ ID NO: 150)
GGTTCACCTTATGAACCCCGAGTATGTCCCCAAACC
GTGCTGTGCGCCAACTAAGCTAAATGCCATCTCGGTT
CTTTACTTTGATGACAACTCCAATGTCATTCTGAAAA
AATACAGGAATATGGTTGTAAGAGCTTGTGGATGCC AC (SEQ ID NO: 149)
[0187] Protein Expression and Purification. The activin/BMP-2, 1b
chimeras, and BMP-2.sub.ma chimeras were expressed using a typical
E. coli expression system, and all 32 chimeras were found in the
inclusion body fractions. The expressed inclusion bodies were
isolated, purified, and refolded. The refolded ligands were
purified using a Hi-trap heparin column (GE Healthcare) and
reversed phase chromatography (GraceVydac). The ligands were
lyophilized and re-suspended in 4 mM HCl, pH 1 for use in all cell
based assays or 10 mM Na acetate, pH 4 for all biophysical assays.
Activin-(3A was expressed in a stably transfected CHO cell line and
purified using techniques known in the art. Noggin was expressed
and purified based on previously described protocols.
[0188] The activin/BMP-2 chimera inclusion bodies were seen as
single bands on a reduced, SDS-PAGE gel and found at the expected
size of .about.13 kDa (FIG. 1a). To standardize the refoldings, all
activin/BMP-2 chimeras were refolded in 100 mL volumes at a
concentration of 50 mg/L. The concentration was chosen based on
previously successful BMP-2, BMP-3, and GDF-5 refoldings. The
volume was picked so that any dimer yield of 2% or greater would
generate enough protein for biophysical activity assays, yet small
enough to be manageable with the large number of samples. Following
refolding, the activin/BMP-2 samples were analyzed for the
formation of pure dimer, the desired product, after elution from
Heparin column (FIG. 1b and c). Surprisingly, all 32 activin/BMP-2
samples showed the presence of some dimer and the chimeras were
ranked based on their refolding efficiency (dimer yield) and
grouped into 4 categories, from poor (<1%, -) to wild type
(>10%, +++) (Table 2). To be classified as a `successful`
chimera, the ligand needed to have a refolding efficiency equal to
or greater than 5%. This efficiency would yield 2.5 mg/L of dimeric
protein from a standard 1 L refolding at 50 mg/L concentration, and
would be considered suitable for experiments where large quantities
are required, such as x-ray crystallography. When refolding
efficiency was calculated, 24 out 32 (75%) of the activin/BMP-2
chimeras met this criteria (Table 2, supplemental, ++ or +++).
TABLE-US-00004 TABLE 2 Dimer Construct Name Yield Rating Rating
System 1b2b3a4a5a6a AB2001 5% ++ +++(wt) >10% 1b2b3a4a5b6a
AB2002 7% ++ ++ 5-9% 1b2a3a4a5b6a AB2003 1% - + 2-4% 1b2a3b4b5a6a
AB2004 9% ++ - <1% 1b2b3b4b5b6b AB2005 >10% +++ 1b2a3a4a5b6b
AB2006 9% ++ 1b2a3a4a5a6b AB2007 >10% +++ 1b2a3a4a5a6a AB2008
>10% +++ 1b2a3a4a5a6a AB2009 ~4% + L66V/V67I 1b(1a_II)2a3a4a5a6a
AB2010 3% + 1b2b3b4b5a6a AB2011 >10% +++ 1b2b3b4b5b6a AB2012
>10% +++ 1b2b3b4b5a6b AB2013 >10% +++ 1b2a3b4b5a6b AB2014
>10% +++ 1b2a3b4b5b6a AB2015 >10% +++ 1b2a3b4b5b6b AB2016
>10% +++ 1b2b3b4a5a6a AB2017 5% ++ 1b2b3b4a5b6b AB2018 2% +
1b2b3b4a5a6b AB2019 3% + 1b2b3b4a5b6a AB2020 3% + 1b2b3a4a5a6b
AB2021 6% ++ 1b2b3a4a5b6b AB2022 5% ++ 1b2b3a4b5b6b AB2023 3% +
1b2b3a4b5b6a AB2024 6% ++ 1b2b3a4b5a6a AB2025 4% + 1b2b3a4b5a6b
AB2026 5% ++ 1b2a3a4b5b6b AB2027 >10% +++ 1b2a3a4b5b6a AB2028 4%
+ 1b2a3a4b5a6b AB2029 1% - 1b2a3a4b5a6a AB2030 2% + 1b2a3b4a5a6a
AB2031 4% + 1b2a3b4a5b6a AB2032 4% + 1b2a3b4a5b6b AB2033 4% +
1b2a3b4a5a6b AB2034 1% -
[0189] To be considered a successful ligand, the activin/BMP-2
chimeras not only have to be refoldable but they also need to
display signaling characteristics. To test for these properties,
all activin/BMP-2 chimeras, regardless of refolding efficiency,
were initially subjected to activin activity assays. Activin-like
signaling characteristics were tested using a whole cell luciferase
reporter assay sensitive to Smad-2/3 activation (as described
below). Activin-.beta.A is known to signal through and activate the
Smad-2/3 pathway, so if any of the activin/BMP-2 chimeras mimic
activin-.beta.A functionality, they should signal in a similar
manner. Out of all 32 chimeras, only 1, 1b2a3a4a5a6a (AB2-008),
signaled in an activin-like manner. AB2-008 activates the
luciferase reporter in a dose dependent manner similar to
activin-.beta.A. When the potency of the AB2-008 chimera was
determined, the EC.sub.50 was calculated to be 64.5 pM. This value
is .about.2 fold lower than activin-.beta.A with an EC.sub.50 of
28.8 pM. To confirm that the luciferase results were a direct
response to Smad-2 activation, phospho-Smad-2 was tested in the
presence of AB2-008. Similar to activin-.beta.A, the addition of
AB2-008 promotes an increase in phospho-Smad-2 levels. As expected,
AB2-008, along with activin-.beta.A, does not stimulate
phospho-Smad-1 production, indicating only activation of a specific
signaling pathway. Interestingly, AB2-008 exhibits BMP-2.sub.ma
refolding efficiency with >10% dimer yield (Table 2).
[0190] To fully test if AB2-008 possesses complete activin-.beta.A
functionality, additional biophysical assays were performed. Cripto
is a known co-receptor for many of the TGF-.beta. ligands and
elicits a wide range of responses. For instance, the presence of
Cripto is required for proper Nodal signaling, while it antagonizes
TGF-.beta.1 and activin signaling. Therefore, using the Smad-2/3
luciferase assay activin-.beta.A and AB2-008 signaling were
monitored in the presence or absence of Cripto. In the presence of
Cripto, activin-.beta.A signaling is decreased by .about.43%
compared activin-.beta.A alone. The AB2-008 chimera exhibits a
similar decrease in signaling of .about.38% when Cripto is added to
the assay. This result confirms that the AB2-008 chimera is a fully
functioning activin mimic by being able to activate the activin
signaling pathway as well having the ability to interact with other
known activin binding partners.
[0191] Based on the results of AB2-008, 2 additional activin/BMP-2
chimeras were generated to see if the potency of the chimeras could
be increased to wild type activin-.beta.A levels. The first
chimera, named 1b2a3a4a5a6a L66V/V67I (AB2-009), introduced a
valine and iso-leucine into the chimera. These residues are found
in the wild type sequence of activin-.beta.A and were originally
mutated to the corresponding BMP-2 residues due to experimental
design constraints (FIG. 7a). The second chimera, named
1b(1a_II)2a3a4a5a6a (AB2-010), replaces the second half of the 1b
section with the corresponding sequence from activin (FIG. 7a).
This leaves only the 13 N-terminal residues preceding the first
structurally conserved cysteine, Leu-66, and Val-67 as components
from BMP-2 in this chimera construct. It is predicted that the
introduction of additional activin residues into AB2-008 will
improve its functional characteristics (i.e. potency). AB2-009 and
AB2-010 were expressed and refolded as previously described for the
other activin/BMP-2 chimeras. Unexpectedly, both of these new
chimeras exhibited decreased refolding efficiency compared to
AB2-008. AB2-009 and AB2-010 saw a decrease from >10% dimer
yield to .about.4% and .about.3% for AB2-009 and AB2-010,
respectively (Table 2). While this result may not be surprising for
AB2-010 since an 11 residue section was mutated, the drastic
decrease for AB2-009 was unexpected. Both the L66V and V67I
mutations are very conservative changes with only a 1 carbon
difference between the side chains of the mutated residues.
[0192] Following refolding, the new chimeras were subjected to the
same Smad-2/3 luciferase assay as AB2-008 previously. AB2-009
activated the reporter in a dose dependent manner and displayed
activity comparable to AB2-008 with an EC.sub.50 of 79.4 pM.
However, while AB2-010 also activated the reporter, it showed a
significant decrease in activity with an EC.sub.50 of 198.6 pM, or
.about.3-fold weaker than AB2-008 and .about.7-fold weaker than
activin-.beta.A. As with AB2-008, both AB2-009 and AB2-0010 showed
Smad-2 phosphorylation. Since AB2-009 and AB2-010 did not show
enhanced signaling characteristics from AB2-008 in the luciferase
assay, they were not subjected to Cripto binding assay.
[0193] While the Smad-2/3 luciferase, Smad-2 phosphorylation, and
Cripto reporter assays indicate that AB2-008, AB2-009, and AB2-010
signal through the activin pathway and function very similarly to
activin-.beta.A, these assays only show function in an in vitro
setting. Therefore, more physiologically relevant experiments are
required to prove that these activin/BMP-2 chimeras will elicit a
biological response similar activin-.beta.A. One classical method
used to test for proper activin function is a follicle stimulating
hormone (FSH) release assay. Rat anterior pituitary cells are known
to release FSH in response to the presence of activin in both in
vivo and in vitro experiments. Therefore, rat anterior pituitary
cells were exposed to increasing amounts of activin-.beta.A or the
activin/BMP-2 chimeras and FSH release was measured by
radioimmunoassay. All three activin/BMP-2 chimeras showed a dose
dependent increase in FSH release similar to activin-.beta.A. The
amount of FSH release stimulated by the chimeras was decreased in
the presence of increasing amounts of Inhibin. Combined with the in
vitro assay results, the FSH release assay confirms that AB2-008,
AB2-009, and AB2-010 possess the complete activin-.beta.A
functional characteristics.
[0194] The chimeras were also tested to check for any additional
signaling properties. BMP-2 is already used as a therapeutic agent
for certain bone treatments and having chimeras with altered BMP-2
function may prove beneficial. To test if any of the activin/BMP-2
chimeras displayed unique signaling characteristics, a similar
experiment to the activin-.beta.A functional assay was performed.
Here, a whole cell luciferase reporter assay sensitive to Smad-1/5
activation, the known BMP-2 signaling pathway, was used rather than
a reporter sensitive to Smad-2/3 activation. Monitoring the
luciferase response in a dose dependant manner, a number of
activin/BMP-2 chimeras exhibit interesting traits. These
activin/BMP-2 chimeras were identified and classified into 3
groups: Those with upregulated or `super` BMP-2 activity; those
with insensitivity to Noggin, a BMP-2 antagonist; or those with
both `super` BMP-2 activity and insensitivity to Noggin.
Activin/BMP-2 chimeras 1b2a3b4b5a6a (AB2-004), 1b2b3b4b5a6a
(AB2-011), 1b2b3b4b5b6a (AB2-012), and 1b2a3b4b5b6a (AB2-015) all
fall into category of enhanced BMP-2 activity with Noggin
insensitivity. In the Smad-1 luciferase assay, these ligands
activate the reporter at the same level as BMP-2.sub.wt using
10.times. less protein (i.e., 10-fold higher activity). Grouped
into the category of upregulated BMP-2 activity is 1b2b3b4b5a6b
(AB2-013). This chimera shows the same 10-fold increase in activity
as AB2-004, -011, -012, -015, but the signal is decreased down to
background levels upon the addition of Noggin, similar to
BMP-2.sub.wt. Chimera, 1b2a3b4b5a6b (AB2-014), fell into the final
category of ligands with normal BMP-2 signaling but with
insensitivity. AB2-014 activates the luciferase reporter to the
same level as BMP-2.sub.wt but its signal cannot be blocked by the
addition Noggin. AB2-008 was also tested to see if it activated the
Smad-1 pathway in addition to activating the Smad-2 pathway.
AB2-008 did not show any Smad-1 activation, even up to levels of 1
.mu.g/ml. This result confirms that AB2-008 is a specific activin
mimic and does not exhibit non-specific signaling
characteristics.
[0195] With the success of the AB2-008 chimera which refolds
efficiently and possesses activin-like signaling characteristics,
the 1b section was examined as a general tool to improve the
refolding of other currently non-refoldable TGF-.beta. ligands. As
mentioned before, the 1b section is 30 a. a. long and comprises the
N-terminus of BMP-2 as well as the residues forming the first beta
strand of finger 1 (FIG. 7a). Based on analysis of the ternary
structure of BMP-2/BMPRIa/ActRII, the majority of the residues
found in section 1b do not form any contacts with either the Type I
or Type II receptors. Indeed, of the few residues which do generate
contacts with the Type I receptor, Val-26, Gly-27, and Trp-28, the
tryptophan is invariant throughout the entire TGF-.beta.
superfamily, while the Gly-27 participates in a backbone
interaction, and the valine is predominantly a non-polar amino acid
at this position throughout the TGF-.beta. superfamily. Based on
this, it is possible that the 1b region, while not critical for
contributing to ligand-receptor affinity and specificity, is very
helpful in proper disulfide bond formation during the chemical
refolding process. Therefore, the 1b section was cloned into the
additional TGF-.beta. ligands BMP-7, BMP-9, and GDF-8. As with the
activin/BMP-2 chimeras, the 1b chimeras were expressed in an E.
coli expression system and the inclusion bodies isolated to high
purity.
[0196] Smad-1 Luciferase Assays in C2C12 Cells.
[0197] Smad1-dependent luciferase assays were performed using
techniques known in the art. In brief, C2C12 myoblast cells are
cultured in Dulbecco's minimum essential medium (DMEM)+5% FBS
supplemented with L-Glutamine and antibiotics. For luciferase
reporter assays, cells were trypsinized, washed twice with PBS and
plated into 48-well plates with DMEM+0.1% FBS. Twenty four hours
later, cells were transfected with -1147Id1-luciferase construct
containing the Smad binding sites (Id1-Luc), a Smad1 expression
construct, and a CAGGS-LacZ plasmid by using Fugene6 (Roche)
according to the manufacturer's instruction and cells were
stimulated with increasing amounts of BMP-2.sub.ma or the various
activin/BMP-2 chimeras added 24 hours post transfection. Luciferase
activity was measured 24 hours after stimulation with ligands and
the values were normalized for transfection efficiency by using
beta-galactosidase activity. The activity of the luciferase
reporter is expressed in fold-induction relative to control values
that are obtained by using -927Id1-luciferase that lacks Smad
binding domains (Id1-Luc mut). To test for the ability of Noggin to
attenuate the Smad1 signaling of the ligands, the luciferase assays
were repeated as described above, with a set dose of Noggin
included in the assay.
[0198] Smad-2 Luciferase Assays in HEK293 Cells.
[0199] HEK293T cells were seeded into 24-well plates coated with
polylysine at a density of 150,000 cells/well. After 24 h cells
were transfected overnight with a mixture of A3 Lux (25 ng) and
.beta.-galactosidase (25 ng) reporter plasmids, the transcription
factor FAST2 (50 ng), and empty pCDNA3 vector (400 ng) using
Perfecting transfection reagent (GenLantis) according to the
manufacturer's recommendations. Then the cells were treated with
increasing doses of activin-.beta.A or activin/BMP-2 chimeras for
16-24 h. The cells were harvested in ice-cold lysis buffer (1%
Triton X-100 in 25 mM glycylglycine, 4 nM EGTA, 15 mM MgSO.sub.4
containing 1 mM dithiothreitol) and assayed for luciferase and
.beta.-galactosidase activities using standard methods. To assess
the ability of the activin/BMP-2 chimeras to bind known TGF-.beta.
co-receptors, the HEK293T cells were treated with increasing doses
of activin-.beta.A or activin/BMP-2 chimeras in the presence or
absence of transfected Cripto for 16-24 h (mouse Cripto construct
was a generous gift from Malcolm Whitman (Department of Cell
Biology, Harvard Medical School, Boston, Mass.)). Activity was then
measures as previously described.
[0200] Follicle Stimulating Hormone (FSH) Release from Rat Interior
Pituitary Cells.
[0201] The assay was performed as previously described in the art.
Briefly, freshly isolated cells from male Sprague-Dawley rat
interior pituitaries from several animals were combined and seeded
into 96-well plates at a density of 50,000 cells/well in .beta.PJ
medium supplemented with 2% fetal bovine serum and appropriate
growth factors. After 24 h cells were treated with increasing doses
of activin-.beta.A or activin/BMP-2 chimeras (0-40 nM). After 72 h,
media were harvested and the concentration of the secreted FSH was
determined by radioimmunoassay.
[0202] Surface Plasmon Resonance (BIAcore) Affinity Studies.
[0203] The affinity of the ligands to BMPRIa, ActRII, and ActRIIb
was monitored by using a Biacore 3000 (GE Healthcare) and the data
were analyzed by using BlAevaluation software ver. 4.1 (GE
Healthcare). Using primary amine coupling, receptor ECDs were
immobilized on a CM5 chip. The receptors were immobilized
independently on flow cells 2-4 for 10 minutes at a flow rate of 5
.mu.L/min and a concentration of 20 .mu.M in 10 mM Na acetate, pH
4.0. Flow cell 1 was left blank, no immobilized protein, as a
negative control. The experiments were performed at a flow rate of
50 .mu.L/min in 20 mM Tris-HCl pH 7.9, 250 mM NaCl, 0.36%
3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),
and 0.005% Tween-20. A minimum of five concentrations, plus a zero
concentration, were run per sample for kinetic analysis and the
data were fit by using a global 1:1 Langmuir binding with mass
transfer.
Example 2
Synthesis of the BMP Heterodimer Ligand
[0204] The crystal structure of BMP2-BMPRIa-ActRIIb has shown that
each receptor molecule do not associate extracellularly and have 4
distinct ligand-receptor interface. This suggested that a
heterodimer would have 2 distinct type I interfaces and 2 distinct
type II interfaces. To characterize functional and other aspects of
the BMP ligand recombinant heterodimers were synthesized. The
purity of the protein was verified by sodium dodecyl
sulfate-polyacrlamide gel electrophoresis (SDS-PAGE). FIG. 1a
illustrates the migration of the BMP2/BMP6 heterodimer as a single
band under non-reducing (lane 1) and as two distinct bands under
reducing conditions (lane 3) on SDS-PAGE. The two distinct bands
correlate to the two different monomer species (about 13 and 15 kDa
respectively), of the BMP2/BMP6 heterodimer. This evidence is
further supported with surface-enhanced laser desorption/ionization
time-of-flight mass spectrometry (SELDI-TOF-MS) data. Three
separate, purified samples of the BMP2 and BMP6 homodimers and the
BMP2/BMP6 heterodimer were assayed on SELDI-TOF-MS. As FIG. 1b
demonstrates, the three samples each correspond to their predicted
mass with no other contaminating species. These assays indicate
that a pure BMP2/BMP6 heterodimer was generated.
[0205] EMP Heterodimer Activity In Vitro.
[0206] To assay the interactions between the BMP2/BMP6 heterodimer
and type I and type II TGF-beta receptor ECDs, surface plasmon
resonance was utilized to measure the in vitro affinity. The
TGF-beta receptors were immobilized to a chip and the TGF-beta
ligands were flowed over the surface while monitoring the
interactions. Table 3 summarizes the ligands tested and the varying
affinities for the type I and type II receptor ECDs. In the case of
the BMP2/BMP6 heterodimer, it adopts the greater affinity from each
of its BMP2 and BMP6 monomer subunits. As shown, the BMP2/BMP6
heterodimer has similar affinity for the type I receptor as the
BMP2 homodimer. However, instead of adopting the type II receptor
ECD affinity from the BMP2 subunit, the BMP2/BMP6 heterodimer has
an affinity similar to the BMP6 homodimer for the type II receptor.
This indicates that the high affinity for the type II receptor ECD
is contributed by the BMP6 monomer subunit while the high affinity
for the type I receptor ECD is contributed by the BMP2 monomer
subunit.
TABLE-US-00005 TABLE 3 Ligand Affinity Data from BIAcore Analysis
Receptor BMPR-Ia Receptor ActRIIb Ligand
k.sub.off[1/s]/k.sub.on[1/M*s] K.sub.D [nM] k.sub.off
[1/s]/k.sub.on[1/M*s] K.sub.D [nM] BMP2 1.11 .times. 10.sup.-3/8.52
.times. 10.sup.5 1.31 2.57 .times. 10.sup.-2/6.68 .times. 10.sup.5
38.5 BMP6 9.37 .times. 10.sup.-3/1.50 .times. 10.sup.5 62.8 1.82
.times. 10.sup.-3/2.73 .times. 10.sup.5 6.68 BMP2/BMP6 1.05 .times.
10.sup.-3/1.03 .times. 10.sup.6 1.02 .sup. 8.58/1.32 .times.
10.sup.9 6.52 Ligand affinity data from BIAcore experiments. The
BIAcore data is shown as the dissociation rate, k.sub.off, and the
association rate, k.sub.on, based on a global fit using the kinetic
model 1:1 Langmuir binding with mass transfer. The binding constant
K.sub.D is calculated as k.sub.off/k.sub.on. The receptors were
immobilized to the chip surface, with the ligands flowed over the
surface.
[0207] To examine if receptor ECD affinity of the BMP2/BMP6
heterodimer correlates with signaling activity, a luciferase
reporter assay was used. Using the C2C12 mouse myoblast cell line,
the BMP ligands were quantitatively tested for the ability to
activate a Smad1 dependent reporter gene. BMP2, BMP6 and BMP2/BMP6,
all showed dose-dependent reporter activation. The BMP2/BMP6
heterodimer ligand showed further greater activation of the
reporter gene than the BMP2 or BMP6 homodimer counterparts (FIG.
2). Up to 22-fold and 400-fold less BMP2/BMP6 was required to
activate the reporter gene to an equivalent level as compared to
the BMP2 and BMP6 homodimers respectively. Given that BMP2/BMP6
heterodimer has a high affinity to both type I and type II receptor
ECDs, the results suggest that increased affinity to receptor
correlates with level of intracellular signaling activity.
[0208] To further characterize activities of BMP2/BMP6 heterodimer,
an ex vivo assay using chick limb bud mesenchyme cells in micromass
culture was used. Primary cultured limb bud mesenchymal cells
undergo chondrogenesis in a BMP-dependent manner, and this system
allows the characterization of homo and heterodimer in a biological
process. FIG. 3 displays micrograph images of the staining of the
chondrogenic nodules whose formation is known to be stimulated by
BMPs. The extent of chondrogenesis is quantitated by measuring dye
bound to the chondrogenic nodule. Similar to the reporter assay, a
dose-dependent activation of chondrogenesis of limb bud mesenchymal
cells by different BMP ligands. A unique aspect of this assay is
that BMP6 has a slightly higher activity than BMP2, while its
activity in the Smad-1-dependent reporter activation was
significantly lower than that of BMP2. This likely involves Type II
receptor-initiated distinct signaling such as the p38 pathway.
Greater activity of BMP2/BMP6 heterodimer to activate
chondrogenesis in this system was observed. BMP2/BMP6 activated
chondrogenesis to the similar level of BMP2 and BMP6 homodimer at
10-fold concentration. The heterodimer ligand also induces a higher
maximum response at the same concentration. This assay allows for
the correlation of not only higher ligand-receptor affinity to
higher signaling activity but extends this observation to an
increase in biological activity.
[0209] The data with ligand-receptor-ECD affinity, in vitro and ex
vivo assays have demonstrated that a functional asymmetric
BMP2/BMP6 dimer was generated. The asymmetric nature of the
BMP2/BMP6 heterodimer allows for the manipulation of specific
TGF-beta receptor sites of the ligand. FIG. 5 displays the specific
mutagenesis of the BMP2/BMP6 heterodimer and the quantification of
the ability of the different mutant variants to activate a Smad1
dependent reporter gene using the same system as described above
(all factors are at 1 nM concentration). The quantified values are
displayed as a percentage fold activation compared to the BMP2/BMP6
wild type heterodimer with no mutagenesis (normalized to 100%
activation of the reporter gene). The BMP2/BMP6 heterodimer ligands
with point mutations to only one of the two type I receptor
interfaces with the two type II receptor interfaces intact (FIG. 5,
samples d and e), are able to activate the reporter gene, although
between 20-80% compared to the BMP2/BMP6 wild type heterodimer. In
contrast, the BMP2/BMP6 heterodimer with point mutations to only
one of the two type II receptor interfaces with the two type I
interfaces intact (FIG. 5, sample f), can not activate the reporter
gene. The BMP2/BMP6 heterodimer ligands with point mutations
disrupting one each of the two type I receptor and type II receptor
interfaces (FIG. 5, samples g and h), cannot activate the reporter
gene. These results demonstrate that 2 type II sites are required
for signaling activity, while only 1 type I site was sufficient for
signaling. The difference of signaling activity between two ligands
having 1 type I site mutation (d and e in FIG. 5) illustrates that
Smad1 activation directly correlates with affinity of a type I site
and type I receptor (Table 3).
[0210] In further studies with the non-signaling BMP2/BMP6
heterodimer mutants, the ability of the ligands to bind receptor
ECD was accessed. Despite the inability of the ligand with only one
active type II receptor site to activate the reporter gene (FIG. 5,
sample f), this ligand is still able to bind type II receptor ECD
under native-PAGE conditions. FIG. 6 illustrates a type II receptor
ECD saturation assay in which the receptor ECD is run on the
native-PAGE both in the absence and presence of ligand. In
comparing the lane with five micrograms of type II receptor ECD and
no factor to the lane with ligand containing two active type II
receptor binding sites, the intensity of the band is diminished by
9.0-fold, indicating that the receptor ECD has been incorporated in
a ligand-receptor complex. This represents about half of the
receptor ECD incorporated into a ligand-receptor complex when
comparing the intensity of three micrograms receptor ECD run alone.
In the next lane, a ligand with only one active type II receptor
site shows an increase in the band intensity by 3.3-fold compared
to the ligand with two active type II receptor sites. And in the
final lane, a ligand with no active type II receptor sites
increases the band intensity by 1.8-fold compared to the ligand
with one active type II receptor site. As expected, the BMP2/BMP6
ligand with one active type II receptor site falls in between the
ligand with two active type II receptor sites and that with no
active type II receptor sites. The single intact type II receptor
interface on the mutated BMP2/BMP6 heterodimer is still able to
bind one type II TGF-beta receptor ECD, indicating that the
extracellular signaling complex can assemble. The inability of the
mutated ligand to signal lies further downstream from signaling
complex assembly at the cell surface. This assay provides proof for
an independent binding model of ligand-receptor complex formation
where a single receptor ECD can bind to one of the four receptor
sites on the ligand regardless of the affinity or functionality of
the other three receptor sites on the ligand.
[0211] The results from the analysis of the recombinant BMP
heterodimers prove that a purified homogeneous heterodimer sample
can be synthesized (FIG. 1). The data further demonstrate that
recombinant BMP heterodimers can be expressed in E. coli as
inclusion bodies, refolded, and purified at a scalable level.
[0212] The data obtained from the surface plasmon resonance
affinity studies (Table 3), shows that a BMP heterodimer is more
potent in vivo and in vitro than the BMP homodimers. As compared to
the homodimer counterparts, the BMP2/BMP6 heterodimer has the
higher affinity receptor sites from each of its covalently linked
monomer subunits. The BMP2/BMP6 heterodimer has a high affinity
type I receptor site comparable with the BMP2 homodimer and a high
affinity type II receptor site comparable with the BMP6 homodimer.
Each of these homodimer ligands secondary receptor sites have lower
affinity for the respective receptor compared to their primary
receptor sites. This heterodimer ligand-receptor affinity data, for
the first time, provides clear evidence for the mechanism of the
high potency TGF-beta heterodimer ligands compared to their
homodimer counterparts. With high affinity for both type I and type
II receptor ECDs, the TGF-beta signaling complex can more readily
assemble and remain assembled as the cell surface. The augmented
affinity of the BMP heterodimer correlates directly to increased
signaling in the whole cell reporter assays (FIG. 2). Additionally,
the ex vivo data from the mesenchyme cell assays (FIGS. 3 and 4)
demonstrates the ability of the BMP2/BMP6 heterodimer to be able to
induce a response at a lower concentration and to a higher maximum
than its homodimer counterparts. This data directly supports the
correlation between increased lignad-receptor affinity, signaling
activity, and biological activity.
[0213] While the high signaling activity of the BMP heterodimer was
readily achieved in the whole cell reporter system (FIG. 2),
elucidating the requirements of the TGF-beta signaling complex and
mechanism of activation proved much more difficult. The BMP
heterodimer constructs with just one type I receptor site and two
type II receptor sites, were still able to activate the reporter
gene, although to a lesser extent compared to the fully functional
BMP heterodimer (FIG. 5). However, the BMP heterodimer constructs
with two type I receptor sites and only one type II receptor site
failed to activate the reporter gene (FIG. 5). This inconsistency
between the number of required active type I and type II receptor
sites on the ligand can not be readily explained. The data
demonstrating complex formation under native-PAGE conditions (FIG.
6), illustrates that the type II receptor ECD can bind to the
mutated heterodimer with only one active type II receptor
interface. This indicates that the problem is not with signaling
complex formation between ligand and receptor ECD at the cell
surface. The complex with only one type II receptor forms as
readily as the complex with one type I receptor, yet the complex
with only one type II receptor does not initiate downstream
signaling.
[0214] The data suggest that a type II receptor kinase
phosphorylates it's partner type II receptor rather than itself,
and such "cross-phosphorylation" is the molecular nature of the
autophosphorylation. Alternatively, physical association of
intracellular kinase domain between two type II receptor is
required for "auto phosphorylation", although ECD does not
associate one another. This initial step of the signaling cascade
cannot occur in the absence of one type II receptor, and thus, no
signal is transduced. The ability of some of the mutant BMP
heterodimer ligands to form an active signaling complex with only
one type I receptor present (FIG. 5), occurs because the two type
II receptor kinases in the signaling complex are able to dimerize,
autophosphorylate, and then transphosphorylate the single type I
receptor kinase. No dimer of the type I receptor kinases is
required because the kinase domain is simply transphosphorylated by
the autophosphorylated dimer of type II receptor kinases.
[0215] The disclosure shows a truly independent ligand-receptor ECD
binding model for the signaling complex formation at the cell
surface. As stated above, mutated BMP2/BMP6 heterodimer ligand with
only one active type II receptor site can still bind a single type
II receptor ECD (FIG. 6). This evidence provides grounds for the
assertion of the signaling complex's ability to form regardless of
the affinity or functionality of the four individual receptor sites
of the ligand. If each receptor site on the ligand is able to bind
in this independent fashion, another layer of complexity is added
to the TGF-beta signaling complex formation. With about forty genes
encoding for ligands and many functional ligands possible because
of heterodimers an intricate signaling mechanism must exist. With
the ability to only signal through twelve receptors, this ligand
driven signaling mechanism must rely on the affinity of ligand's
individual receptor sites to the different receptors. This is only
possible if each of the four receptor sites on the ligand act
independently to recruit a receptor into the signaling complex. In
order to elicit different biological functions through the same
twelve receptors, the independent and individual affinities of each
of the four receptor sites on the ligand is the key factor in fine
tuning the biological response. In this point of view, a role of
specific ligand (homodimer or heterodimer) is to assemble distinct
set of type I and type II receptors with distinct affinity, which
in turn, generate different level of signaling and complexity of
TGF-b signaling.
[0216] The scalable generation of a novel BMP2/BMP6 construct with
high activity in vitro and ex vivo has far reaching implications.
This molecule served as the basis to determine the assembly of the
TGF-beta superfamily ligand-receptor signaling complex and to
demostrate the direct correlation between ligand-receptor affinity,
signaling activity, and biological activity. The differences in
affinity between ligand and receptor are crucial and the asymmetric
heterodimer ligand signaling adds further complexity to the
biological activity of the TGF-beta molecules. the study with the
BMP heteromider illustrates how each ligand-receptor interaction
contributes to the activity of the TGF-beta superfamily.
[0217] Generation of Heterodimer.
[0218] The mature domains of human BMP2 (residues 1-110) and human
BMP6 (residues 1-132) were expressed in E. coli as inclusion
bodies. Mutations to the wild type ligand sequences were based on
previously published findings which disrupt the ligand-receptor
interfaces (Keller et al., 2004; Kirsch et al., 2000). The
expressed inclusion bodies were isolated, purified, and refolded.
The refolded BMP2 and BMP6 homodimers, and BMP2/BMP6 heterodimer
were purified using a HiTrap heparin column (GE Healthcare) and
reversed phase chromatography (GraceVydac). The ligands were
lyophilized and re-suspended in 10 mM sodium acetate pH 4.0. The
ECDs of human BMPRIa (residues 1-129) and mouse ActRIIb (residues
1-98) were expressed in E. coli as thioredoxin fusion proteins.
Mouse ActRII-ECD (residues 1-102) was expressed and purified from a
P. pastoris expression system.
[0219] Surface Plasmon Resonance (BIAcore) Affinity Studies.
[0220] The affinity of the ligands to BMPRIa, ActRII, and ACTRIIb
was monitored by using a Biacore 3000 (GE Healthcare) and the data
were analyzed by using BIAevaluation software ver. 4.1 (GE
Healthcare). Using primary amine coupling, receptor ECDs were
immobilized on a CM5 chip. The receptors were immobilized
independently on flow cells 2-4 for 10 minutes at a flow rate of 5
.mu.L/min and a concentration of 20 .mu.M in 10 mM sodium acetate,
pH 4.0. Flow cell 1 was left blank with no immobilized protein as a
negative control. The experiments were performed at a flow rate of
50 .mu.L/min in 20 mM Tris-HCl, pH 7.9, 250 mM NaCl, 0.36%
3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS),
and 0.005% Tween-20. At least five concentrations, plus a zero
concentration, were run per sample for kinetic analysis and the
data were fit by using a global 1:1 Langmuir binding with mass
transfer.
[0221] Luciferase reporter assays. Smad1-dependent luciferase
assays were performed. In brief, C2C12 cells are cultured in
Dulbecco's minimum essential medium (DMEM)+5% FBS supplemented with
L-glutamine and antibiotics. For luciferase reporter assays, cells
were trypsinized, washed twice with PBS, and plated into 48-well
plates with DMEM+0.1% FBS. Twenty-four hours later, cells were
transfected with -1147Id1-luciferase construct containing the Smad
binding sites (Id1-Luc) (Nakashima et al., 2001), a Smad-1
expression construct, and a CAGGS-LacZ plasmid using Fugene6
(Roche) according to the manufacturer's instruction. Luciferase
activity was measured 24 h after stimulation with ligands, and the
values were normalized for transfection efficiency using
beta-galactosidase activity. The activity of the luciferase
reporter is expressed in fold induction relative to control values
that are obtained using -927Id1-luciferase that lacks Smad1 binding
domains (Id1-Luc mut).
[0222] Chick Limb Bud Micromass Assays.
[0223] Chick embryos at Hamburger-Hamilton stage 23-24 (Hamburger
and Hamilton, 1951) were collected in Hanks solution containing
Ca2+ and Mg2+, and distal 1/3 part of limb buds were dissected out.
Ectodermal sheets were removed by trypsinization (0.5% in Hanks
solution with Ca2+ and Mg2+) on ice for 30 min, and then
mesenchymal tissues were recovered and incubated in Ca.sup.2+ and
Mg.sup.2+-free Hanks solution at 37.degree. C. for 15 min. The
mesenchyme cells were dissociated into single cells by pipetting in
OptiMEM medium (Invitrogen) containing 1% FBS. Cultures were seeded
into 96-well plates at 4.times.10.sup.5 cells/well. After 1 hour,
media containing each ligand was added. Fresh media with ligands
was changed daily, and cells were analyzed for chondrogenesis by
Alcian blue staining to visualize cartilage nodule and
quantification of chondrogenesis as described (Wada et al.,
2003).
[0224] Native-PAGE Ligand-Receptor ECD Complex Formation.
[0225] Five micrograms of purified ActRII-ECD alone and with ten
micrograms of BMP2/BMP6, BMP2/BMP6 with a single active type II
receptor interface, or BMP2 with no active type II receptor
interfaces was loaded onto a native-PAGE gel in 50 mM Tris-HCl, pH
7.9, 700 mM NaCl, and 1.8% CHAPS. The Coomassie Brilliant Blue
(Bio-Rad) stained gel was analyzed using the "Integrated Density"
function with NIH ImageJ software (Abramoff et al., 2004).
Example 3
(1) Development of Stem Cell Media (Valera et al., 2010) Using
AB2-008
[0226] Culturing human embryonic stem cells (hESC) in feeder free
conditions requires the use of complex formulation media to
maintain pluripotency. Unfortunately, commercial media are very
expensive since the growth factors required for the media are
difficult to produce in mammalian cells. We have used mTeSR1
formulation to derive a new medium (CIVA medium or mCIVA) for
culturing human embryonic stem (hES) cells, and deriving and
culturing induced pluripotent stem (iPS) cells (see FIG. 2). CIVA
medium substitutes TGF.beta.1 in mTeSR1 for AB2-008, a new chimeric
protein with similar activity to Activin-A. hES cells cultured in
this medium on matrigel coating maintain pluripotent morphology for
more than 20 passages without karyotypic abnormalities. These cells
are also positive for the pluripotency markers TRA-1-60 and SSEA-4,
and differentiate in response to BMP-2 treatment. iPS cells
cultured in this medium also retain morphological characteristics
of pluripotency and expression of pluripotency markers. This new
CIVA medium is also suitable to derivate iPS cells from human
foreskin fibroblasts. CIVA medium has all the properties desired of
other commercial media for hES cells but can be formulated for
considerably less cost than currently available media. FIG. 2.
Development of mCIVA formulation using H9 hES cell line.
[0227] FIG. 9 shows H9 hES cells cultured in mCIVA using different
concentrations of AB2-008 in the absence or presence of human FGF2.
A. Differentiated H9 cells after 3 passages (1 ng/mL, AB2-008; no
FGF2). B. Differentiated H9 cells after 3 passages (10 ng/mL,
AB2-008; no FGF2). C. Differentiated H9 cells after 11 passages
(100 ng/mL, AB2-008; no FGF2). D. Differentiated H9 cells after 12
passages (1 ng/mL, AB2-008; 100 ng/mL, FGF2). E. H9 cells after 13
passages (10 ng/mL, AB2-008; 100 ng/mL, FGF2). F. Differentiated H9
cells after 9 passages (100 ng/mL, AB2-008; 100 ng/mL, FGF2).
Differentiated cells are denoted by arrows.
(2) Mineralization Data of AB2-004, AB2-011, AB2-015 (Yoon et al.,
2010)
[0228] Von Kossa staining was used to monitor the development of
pre-osteoblast cell line (MC3T3-E1) for extracellular
mineralization by Ca deposition. AB2-004 and AB2-011 show dramatic
increase of the stain of nearly 10 times or more intensity. AB2-015
shows the most increase but past 7 days period (not shown here).
Control group (A) shows no appreciable mineralization, whereas the
control ligand (BMP2) shows modest increase of Ca flux and
deposition as shown in Row B. of FIG. 10.
(3) Adult Rat Regeneration by AB2-004 (in Collaboration) P3 Digit
of Adult Rat was Severed
[0229] Either BMP2 or AB2-004 soaked in an agarose gel bead were
added at the sight of sugery. Bone regrowth was monitored.
BMP2-treated tissue shows no bone recovery. AB2-004-treated digit
shows full recovery.
(4) Smad2-Based Signaling (Luciferase) Assay by AB2-008, AB2-009,
AB2-010 (Allendorph et al., 2010)
[0230] AB2-008, AB2-009, and AB2-010 activate the Smad2 pathway in
a manner nearly undistinguishable from activin-.beta.A. Potency for
the chimeras is slightly reduced compared to activin-.beta.A, from
5 to 20-fold (see FIG. 11).
(5) Phospho-Smad-2 Assays by AB2-008, AB2-009, and AB2-010
[0231] Activin-.beta.A specifically phosphorylates Smad-2 and not
Smad-1. This is in contrast to BMP-2 where we see specific
phosphorylation of Smad-1 and not Smad-2. AB2-008, AB2-009, and
AB2-010 display the same Smad-2 phosphorylation pattern as
activin-.beta.A. This confirms all three ligands stimulate the
activin-.beta.A signaling pathway in a manner similar to
activin-.beta.A (see FIG. 11).
(6) Follicle Stimulating Hormone Release by AB2-008, AB2-009, and
AB2-010
[0232] When activin-.beta.A is added to rat interior pituitary
cells, it causes a dose dependent release of Follicle stimulating
hormone (FSH). The activin-.beta.A induced release of FSH can be
blocked by the addition of the antagonist Inhibin. Similar to
activin-.beta.A, AB2-008, AB2-009, and AB2-010 cause the release of
FSH and this stimulation is decreased in the presence of
Inhibin.
(7) Co-Receptor Binding with Cripto by AB2-008
[0233] Addition of Cripto reduces both activin-.beta.A and AB2-008
signaling by comparable levels. These data indicate that AB2-008
possesses ability to bind Cripto, activin-.beta.Aco-receptor,
confirming the functional similarity between AB2-008 and
activin-.beta.A.
(8) AB2-008 and Activin-.beta.A Receptor Affinity
[0234] AB2-008 shows the receptor binding profile similar to that
of activin-.beta.A, which has high affinity to ActRII and none for
BMPRIa. Further, the two ligands have nearly the same binding
affinity. AB2-008 is .about.1.7 weaker than activin-.beta.A.
TABLE-US-00006 TABLE 4 Receptor affinity of Activin-.beta.A versus
AB2-008 Receptor Affinity Data from BIAcore Experiments BMPRIa-ECD
ActRII-ECD Ligand k.sub.off[1/s]/k.sub.on[1/M*s] K.sub.D [nM]
k.sub.off[1/s]/k.sub.on[1/M*s] K.sub.D [nM] Activin-.beta.A No
Binding N.A. 7.16 .times. 10.sup.-4/3.52 .times. 10.sup.6 0.203
AB2-008 No Binding N.A. 8.24 .times. 10.sup.-4/2.39 .times.
10.sup.6 0.344 The receptors were immobilized to the chip surface
with the ligands flowed over the surface. The data were fit to a
kinetic model (1:1 Langmuir binding with mass transfer) in which
K.sub.D is calculated as k.sub.off/k.sub.on. The table reports data
from a single trial. No Binding indicates that the interaction was
not detectable.
(9) Signaling Activity of AB2-011, AB2-012, and AB2-015 by Smad-1
Pathway
[0235] AB2-004, AB2-011, AB2-012, and AB2-015 activate the Smad-1
pathway more potently than BMP-2. This activation is 3 to 8-fold
higher than BMP-2 (FIG. 14).
(10) Receptor Binding Affinity of AB2-004, AB2-011, AB2-012, and
AB2-015
[0236] AB2-004, AB2-011, AB2-012, and AB2-015 show similar binding
affinity to ActRII as activin-.beta.A. This is .about.100 fold
higher than the binding BMP-2 has for the same receptor. The type I
receptor binding for the AB chimeras ranges from near BMP-2 levels
(AB2-004) to activin-.beta.A levels (no binding to BMPRIa for
AB2-015).
TABLE-US-00007 TABLE 5 Receptor binding affinity of AB2-004,
AB2-011, AB2-012, and AB2-015 Receptor Affinity Data from BIAcore
Experiments BMPRIa-ECD ActRII-ECD Ligand
k.sub.off[1/s]/k.sub.on[1/M*s] K.sub.D [nM]
k.sub.off[1/s]/k.sub.on[1/M*s] K.sub.D [nM] BMP-2 7.54 .times.
10.sup.-4/3.60 .times. 10.sup.5 2.09 4.05 .times. 10.sup.-2/1.10
.times. 10.sup.6 36.8 Activin-.beta.A No Binding N.A. 7.16 .times.
10.sup.-4/3.52 .times. 10.sup.6 0.203 AB2-004 1.85 .times.
10.sup.-2/1.13 .times. 10.sup.6 16.4 1.87 .times. 10.sup.-4/4.91
.times. 10.sup.5 0.381 AB2-011 1.93 .times. 10.sup.-2/4.07 .times.
10.sup.5 47.4 3.39 .times. 10.sup.-4/6.46 .times. 10.sup.5 0.525
AB2-015 No Binding N.A. 3.71 .times. 10.sup.-4/1.61 .times.
10.sup.6 0.230 AB2-012 8.48 .times. 10.sup.-2/1.61 .times. 10.sup.5
526 1.60 .times. 10.sup.-3/3.39 .times. 10.sup.6 0.472 The
receptors were immobilized to the chip surface with the ligands
flowed over the surface. The data were fit to a kinetic model (1:1
Langmuir binding with mass transfer) in which K.sub.D is calculated
as k.sub.off/k.sub.on. The table reports data from a single trial.
No Binding indicates that the interaction was not detectable.
(11) Noggin Insensitivity of AB2-004, AB2-011, AB2-012, and
AB2-015
[0237] Noggin suppresses the signaling acitivity by directly
complexing with the ligand, and rendering it unable to bind its own
receptors for signaling. In contrast to BMP-2 which is blocked to
near background levels in the presence of Noggin, the higher
signaling of AB2-004, AB2-011, and AB2-015 are not inhibited by
Noggin. AB2-012 is partially insensitive to Noggin Inhibition, with
a .about.50% decrease in signaling with the addition of Noggin
(FIG. 15). This property makes them particularly powerful in their
cellular signaling ability in vivo, including bone
regeneration.
(12) Production Efficiency
TABLE-US-00008 [0238] TABLE 6 Production efficiency of refolding
from E. coli inclusion body. Construct Dimer Yield Rating
1b2b3b4b5b6a >10% +++ 1b2b3b4b5a6a >10% +++ 1b2b3b4b5a6b
>10 .sup. +++ 1b2b3b4a5a6a 5% +++ 1b2b3b4a5b6b 2% + 1b2b3b4a5a6b
3% + 1b2b3b4a5b6a 3% + 1b2b3a4a5a6a 5% ++ 1b2b3a4a5a6b 6% ++
1b2b3a4a5b6b 5% ++ 1b2b3a4a5b6a 7% ++ 1b2b3a4b5b6b 3% +
1b2b3a4b5b6a 6% ++ 1b2b3a4b5a6a 4% + 1b2b3a4b5a6b 5% ++
1b2a3a4a5a6a >10% +++ 1b2a3a4a5a6b >10% +++ 1b2a3a4a5b6b 9%
++ 1b2a3a4a5b6a >10% +++ 1b2a3a4b5b6b >10% +++ 1b2a3a4b5b6a
4% + 1b2a3a4b5a6b 1% - 1b2a3a4b5a6a 2% + 1b2a3b4b5b6b >10% +++
1b2a3b4b5b6a >10% +++ 1b2a3b4b5a6a 9% ++ 1b2a3b4b5a6b >10%
+++ 1b2a3b4a5a6a 4% + 1b2a3b4a5b6a 4% + 1b2a3b4a5b6b 4% +
1b2a3b4a5a6b 1% - 1b2a3a4a5a6a L66V/V67I 4% + 1b(1a_II)2a3a4a5a6a
3% +
(13) Receptor Binding Affinity of BMP2/BMP6 Heterodimer (Isaacs et
al., 2010)
[0239] BMP2/BMP6 heterodimer has the binding characteristics of
BMP2 for type I receptor BMPRIa and BMP6 for the type II receptor
ActRIIb. Maintaining high affinity for each receptor type by the
heterodimer ligand makes BMP2/BMP6 heterodimer stronger in
signaling activities than its homodimeric couterparts, BMP2 and
BMP6 homodimers (Table 7).
TABLE-US-00009 TABLE 7 Receptor binding affinity measured by
Surface plasmon resonance. Receptor BMPRIa K.sub.D Receptor ActRIIb
K.sub.D Ligand k.sub.off[1/s]/k.sub.on[1/M*s] [nM]
k.sub.off[1/s]/k.sub.on[1/M*s] [nM] BMP2 1.11 .times.
10.sup.-3/8.52 .times. 10.sup.5 1.31 2.57 .times. 10.sup.-2/6.68
.times. 10.sup.5 38.5 BMP6 9.37 .times. 10.sup.-3/1.50 .times.
10.sup.5 62.8 1.82 .times. 10.sup.-3/2.73 .times. 10.sup.5 6.68
BMP2/ 1.05 .times. 10.sup.-3/1.03 .times. 10.sup.6 1.02 8.61
.times. 10.sup.-3/1.32 .times. 10.sup.6 6.52 BMP6
(14) SMAD-1 Signaling Activity of BMP2/BMP6 Heterodimer
[0240] The BMP2/BMP6 heterodimer is much more active than either
BMP2 or BMP6 alone. BMP2/BMP6 has an EC50 that is at least an order
of magnitude higher than BMP2 or BMP6 alone. Further, the maximal
response reached by BMP2/BMP6 is higher than the combination of
maximum signal reached by BMP2 and BMP6 alone.
(15) Chick Limb Bud Micromass assays for BMP2/BMP6 Heterodimer
[0241] BMP2/BMP6 induces chondrogenesis more potently than either
BMP2 or BMP6 homodimers. In chick limb bud mesenchyme cell
micromass culture chrondogenesis assays, after three days we see
that BMP2/BMP6 induces chondrogenesis at both lower concentrations
and to a higher level than either BMP2 or BMP6.
Example 4
Description of Subdomains (Building Blocks) for Generating Designer
Ligand
[0242] In order to create the chimeras, a first step was deciding
where to make the borders for each of the segments. The chimera
library has been constructed using activin-.beta.A and BMP-2 as two
sequence sources. To design the cut-off regions (Junction) for the
sections to make the activin/BMP-2 (AB) chimera, a structure-guided
approach combined with protein sequence alignment was used.
Initially, the 3-dimensional crystal structures of activin-.beta.A
(Harrington et al., 2006) and BMP-2 (Allendorph et al., 2006) were
inspected structurally. From this analysis, the ligands were
loosely divided into 6 distinct sections (see FIG. 7 for segments 1
through 6). The exact segment junctions were ultimately determined
following a protein sequence alignment of the two ligands to
minimize any sequence changes of either protein sequence as a
result of joining the Junction. Further, the segmental boundaries
were chosen to be located in structural regions away from receptor
binding sites.
[0243] Detailed descriptions of Junctions: Between segments 1 and 2
(Junction 1): Focusing on the boundary of segment 1 and segment 2,
we found a 10-residue region that is highly conserved between BMP-2
and activin-.beta.A. Indeed, 8 of the 10 residues are identical and
the other two are very conservative differences. This area is
located in the tip region of Finger 1 and depending of the ligand,
makes or is predicted to make limited contacts with either receptor
type. Based on the ternary crystal structure of BMP-2/BMPRIa/ActRII
(Allendorph et al., 2006), only Val-26, Gly-27, and Trp-28 (BMP-2
numbering) generate contacts with the type I receptor. Of these
three residues, only Val-26 is different between the ligands, but
it is a very conservative change since the corresponding residue in
activin-.beta.A is Ile-23. Since the residues in this region are
very similar and not involved in receptor binding, it makes for a
good boundary point for segment 1 and 2.
[0244] Between segments 2 and 3 (Junction 2): Moving to the
boundary region between segments 2 and 3, another good area for our
boundary cut-off can be found. Here, a 4-residue sequence that is
identical between activin-.beta.A and BMP-2 exists. When the
ligands are properly folded, this region is located in the center
of the dimer, with both cyteines participating in the cystine knot.
This is advantageous because the residues here are buried from the
surface of the ligands and do not participate in any
ligand-receptor interactions.
[0245] Between segments 4 and 5 (Junction 4): Similar to the
segment 2/3 boundary, the segment 4/5 boundary is situated in an
excellent location for the cut-off. Here, we find a 5-residue
region of sequence identity and, as with the segment 2/3 boundary,
this region is buried at the center of the ligand dimer. The 2
cysteine residues participate in both the cystine knot as well as
the inter-monomer disulfide bond. Again, this location prevents the
residues in this region from participating in receptor binding
interactions.
[0246] Between segments 5 and 6 (Junction 5): To extend the design
of BMP-2 and activin-.beta.A chimeras, other boundary regions have
been chosen to facilitate generating RASCH constructs using all
members of the TGF-.beta. superfamily. Along with sharing
structural architecture, the TGF-.beta. superfamily ligands seem to
have certain regions in their protein sequences that are highly
conserved. Interestingly, these regions coincide with the boundary
regions chosen for making the BMP-2 and activin-.beta.A chimeras.
For example, in the boundary region of 4 and 5, most ligands share
3 out of the 4 residues that define the boundary domain. This high
degree of similarity, coupled with these regions being isolated
from the receptor binding sites, indicates RASCH as the universal
strategy to create a library of Designer Ligands with new
functionalities.
[0247] Between segments 3 and 4 ((Junction 3): The boundary between
segments 3 and 4 is subject to structural variability between
different subfamilies, in which ligand-receptor assembly mechanism
can differ substantially. In that regard, segments 3 and 4 can be
treated as one segmental piece such that two segments will be
derived from the common parental strand to preserve their
structural integrity.
[0248] The structural similarity among all TGF-beta superfamily
ligands forms the rational basis for designing chimeric protein by
exchanging (swapping) related segments of the sequences known to
carry out certain functionality such as molecular recognition.
Protein engineering of Antibody chain, or more specifically of
antibody fragment (Fab), will be a prime example where the basic
structural scaffold is built on the Core architecture of the light-
and heavy chain sequences, for which six variable loops, three from
each of the two chains, are responsible for the role of
epitope-binding specificity. In the similar vein, the TGF-beta
superfamily ligands share their structural framework as a
butterfly-like architecture. A portion(s) of the sequence segments
functionally equivalent to variable loop regions of Antibody can
then be `implanted` to transfer recognition specificity from one
ligand to another. Our design principle distinguishes itself from
the aforementioned `functional transfer by sequence implantation`.
The new chimeric library is created on the basis of structural
feasibility of each subdomain as defined by each Junction. The
junctions between the various domains of the TGF-beta family
members used to generate the chimeras of the disclosure provide
useful building blocks of the chimera library. By this reasoning,
Junctions 1, 2, 4, and 5 are well defined to be broadly applicable
to all TGF-beta superfamily members, whereas Junction 3 is not
broadly applicable. The application of Junction 3 in the chimera
design depends on the target sequences, in which case subdomain
segments 3 and 4 can be treated instead as one segment in designing
the chimera library. The approach maximizes the chance of producing
such protein products that are foldable, for which functional
characterization will then follow.
[0249] Table of additional sequences:
TABLE-US-00010 Protein TGF-.beta. Ligand DNA Sequence Sequence
BMP-2 caagccaaacacaaacagcggaaacgccttaagtccagctgta
QAKHKQRKRLKSSCKRHPL agagacaccctttgtacgtggacttcagtgacgtggggtggaat
YVDFSDVGWNDWIVAPPG gactggattgtggctcccccggggtatcacgccttttactgccac
YHAFYCHGECPFPLADHLN ggagaatgcccttttcctctggctgatcatctgaactccactaatc
STNHAIVQTLVNSVNSKIPK
atgccattgttcagacgttggtcaactctgttaactctaagattcct
ACCVPTELSAISMLYLDENE aaggcatgctgtgtcccgacagaactcagtgctatctcgatgct
KVVLKNYQDMVVEGCGCR gtaccttgacgagaatgaaaaggttgtattaaagaactatcagga
(SEQ ID NO: 2) catggttgtggagggttgtgggtgtcgc (SEQ ID NO: 1) BMP-3
cagtggattgaacctcggaattgcgccaggagatacctcaaggt QWIEPRNCARRYLKVDFAD
(osteogenin) agactttgcagatattggctggagtgaatggattatctcccccaa
IGWSEWIISPKSFDAYYCSG
gtcctttgatgcctattattgctctggagcatgccagttccccatgc
ACQFPMPKSLKPSNHATIQS caaagtctttgaagccatcaaatcatgctaccatccagagtatag
IVRAVGVVPGIPEPCCVPEK tgagagctgtgggggtcgttcctgggattcctgagccttgctgtg
MSSLSILFFDENKNVVLKV taccagaaaagatgtcctcactcagtattttattctttgatgaaaat
YPNMTVESCACR aagaatgtagtgcttaaagtataccctaacatgacagtagagtctt (SEQ ID
NO: 43) gcgcttgcaga (SEQ ID NO: 42) BMP-4(BMP-2
agccctaagcatcactcacagcgggccaggaagaagaataag SPKHHSQRARKKNKNCRR b)
aactgccggcgccactcgctctatgtggacttcagcgatgtggg HSLYVDFSDVGWNDWIVA
ctggaatgactggattgtggccccaccaggctaccaggccttct PPGYQAFYCHGDCPFPLAD
actgccatggggactgcccctttccactggctgaccacctcaac HLNSTNHAIVQTLVNSVNS
tcaaccaaccatgccattgtgcagaccctggtcaattctgtcaatt SIPKACCVPTELSAISMLYL
ccagtatccccaaagcctgttgtgtgcccactgaactgagtgcc DEYDKVVLKNYQEMVVEG
atctccatgctgtacctggatgagtagataaggtggtactgaaaa CGCR
attatcaggagatggtagtagagggatgtgggtgccgc (SEQ (SEQ ID NO: 45) ID NO:
44) BMP-5 gcagccaacaaacgaaaaaatcaaaaccgcaataaatccagct
AANQNRNKSSSHQDSSRMS ctcatcaggactcctccagaatgtccagtgttggagattataaca
SVGDYNTSEQKQACKKHEL caagtgagcaaaaacaagcctgtaagaagcacgaactctatgt
YVSFRDLGWQDWIIAPEGY gagcttccgggatctgggatggcaggactggattatagcacca
AAFYCDGECSFPLNAHMNA
gaaggatacgctgcattttattgtgatggagaatgttcttttccactt
TNHAIVQTLVHLMFPDHVP aacgcccatatgaatgccaccaaccacgctatagttcagactct
KPCCAPTKLNAISVLYFDDS
ggttcatctgatgtttcctgaccacgtaccaaagccttgttgtgctc SNVILKKYRNMVVRSCGCH
caaccaaattaaatgccatctctgttctgtactttgatgacagctcc (SEQ ID NO: 47)
aatgtcattttgaaaaaatatagaaatatggtagtacgctcatgtg gctgccac(SEQ ID NO:
46) BMP-6(Vgr-1) caacagagtcgtaatcgctctacccagtcccaggacgtggcgc
QQSRNRSTQSQDVARVSSA gggtctccagtgcttcagattacaacagcagtgaattgaaaaca
SDYNSSELKTACRKHELYV gcctgcaggaagcatgagctgtatgtgagtttccaagacctggg
SFQDLGWQDWIIAPKGYAA atggcaggactggatcattgcacccaagggctatgctgccaatt
NYCDGECSFPLNAHMNAT actgtgatggagaatgctccttcccactcaacgcacacatgaat
NHAIVQTLVHLMNPEYVPK gcaaccaaccacgcgattgtgcagaccttggttcaccttatgaa
PCCAPTKLNAISVLYFDDNS ccccgagtatgtccccaaaccgtgctgtgcgccaactaagctaa
NVILKKYRNMVVRACGCH atgccatctcggttctttactttgatgacaactccaatgtcattctga
(SEQ ID NO: 49) aaaaatacaggaatatggttgtaagagcttgtggatgccac (SEQ ID
NO: 48) BMP-7(OP-1) tccacggggagcaaacagcgcagccagaaccgctccaagac
STGSKQRSQNRSKTPKNQE gcccaagaaccaggaagccctgcggatggccaacgtggcag
ALRMANVAENSSSDQRQA agaacagcagcagcgaccagaggcaggcctgtaagaagcac
CKKHELYVSFRDLGWQDW gagctgtatgtcagcttccgagacctgggctggcaggactggat
IIAPEGYAAYYCEGECAFPL catcgcgcctgaaggctacgccgcctactactgtgagggggag
NSYMNATNHAIVQTLVHFI tgtgccttccctctgaactcctacatgaacgccaccaaccacgc
NPETVPKPCCAPTQLNAISV catcgtgcagacgctggtccacttcatcaacccggaaacggtg
LYFDDSSNVILKKYRNMVV cccaagccctgctgtgcgcccacgcagctcaatgccatctccgt
RACGCH cctctacttcgatgacagctccaacgtcatcctgaagaaatacag (SEQ ID NO:
51) aaacatggtggtccgggcctgtggctgccac (SEQ ID NO: 50) BMP-8(OP2)
gcagtgaggccactgaggaggaggcagccgaagaaaagcaa AVRPLRRRQPKKSNELPQA
cgagctgccgcaggccaaccgactcccagggatctttgatgac NRLPGIFDDVHGSHGRQVC
gtccacggctcccacggccggcaggtctgccgtcggcacgag RRHELYVSFQDLGWLDWVI
ctctacgtcagcttccaggacctcggctggctggactgggtcat APQGYSAYYCEGECSFPLD
cgctccccaaggctactcggcctattactgtgagggggagtgct SCMNATNHA1LQSLVHLMK
ccttcccactggactcctgcatgaatgccaccaaccacgccatc PNAVPKACCAPTKLSATSV
ctgcagtccctggtgcacctgatgatgccagacgcagtcccca LYYDSSNNVILRKHRNMVV
aggcgtgctgtgcacccaccaagctgagcgccacctctgtgct KACGCH
ctactatgacagcagcaacaatgtcatcctgcgcaagcaccgc (SEQ ID NO: 53)
aacatggtggtcaaggcctgcggctgccac (SEQ ID NO: 52) BMP-9(GDF-2)
agcgccggggctggcagccactgtcaaaagacctccctgcgg SAGAGSHCQKTSLRVNFED
gtaaacttcgaggacatcggctgggacagctggatcattgcacc IGWDSWIIAPKEYEAYECK
caaggagtatgaagcctacgagtgtaagggcggctgcttcttcc GGCFFPLADDVTPTKHAIV
ccttggctgacgatgtgacgccgacgaaacacgctatcgtgca QTLVHLKFPTKVGKACCVP
gaccctggtgcatctcaagttccccacaaaggtgggcaaggcc TKLSPISVLYKDDMGVPTL
tgctgtgtgcccaccaaactgagccccatctccgtcctctacaag KYHYEGMSVAECGCR
gatgacatgggggtgcccaccctcaagtaccattacgagggca (SEQ ID NO: 55)
tgagcgtggcagagtgtgggtgcaggtag (SEQ ID NO: 54) BMP-10
aacgccaaaggaaactactgtaagaggaccccgctctacatcg NAKGNYCKRTPLYIDFKEIG
acttcaaggagattgggtgggactcctggatcatcgctccgcct WDSWIIAPPGYEAYECRGV
ggatacgaagcctatgaatgccgtggtgtttgtaactaccccctg CNYPLAEHLTPTKHAIIQAL
gcagagcatctcacacccacaaagcatgcaattatccaggcctt VHLKNSQKASKACCVPTKL
ggtccacctcaagaattcccagaaagcttccaaagcctgctgtg EPISILYLDKGVVTYKFKYE
tgcccacaaagctagagcccatctccatcctctatttagacaaag GMAVSECGCR
gcgtcgtcacctacaagtttaaatacgaaggcatggccgtctcc (SEQ ID NO: 57)
gaatgtggctgtaga (SEQ ID NO: 56) BMP-15(GDF-
caagcagatggtatctcagctgaggttactgcctcttcctcaaaa QADGISAEVTASSSKHSGPE
9b) catagcgggcctgaaaataaccagtgttccctccaccctttccaa
NNQCSLHPFQISFRQLGWD atcagcttccgccagctgggttgggatcactggatcattgctccc
HWIIAPPFYTPNYCKGTCLR cctttctacaccccaaactactgtaaaggaacttgtctccgagtac
VLRDGLNSPNHAIIQNLINQ tacgcgatggtctcaattcccccaatcacgccattattcagaacct
LVDQSVPRPSCVPYKYVPIS tatcaatcagttggtggaccagagtgtcccccggccctcctgtgt
VLMIEANGSILYKEYEGMIA
cccgtataagtatgttccaattagtgtccttatgattgaggcaaatg ESCTCR
ggagtattttgtacaaggagtatgagggtat (SEQ ID NO: 59)
gattgctgagtcttgtacatgcaga (SEQ ID NO: 58) GDF-1
gacgccgaacccgtgttgggcggcggccccgggggcgcttgt DAEPVLGGGPGGACRARRL
cgcgcgcggcggctgtacgtgagcttccgcgaggtgggctgg YVSFREVGWHRWVIAPRGF
caccgctgggtcatcgcgccgcgcggcttcctggccaactact LANYCQGQCALPVALSGSG
gccagggtcagtgcgcgctgcccgtcgcgctgtcggggtccg GPPALNHAVLRALMHAAA
gggggccgccggcgctcaaccacgctgtgctgcgcgcgctca PGAADLPCCVPARLSPISVL
tgcacgcggccgccccgggagccgccgacctgccctgctgc FFDNSDNVVLRQYEDMVV
gtgcccgcgcgcctgtcgcccatctccgtgctcttctttgacaac DECGC
agcgacaacgtggtgctgcggcagtatgaggacatggtggtg (SEQ ID NO: 61)
gacgagtgcggctgccgc (SEQ ID NO: 60) GDF-3(Vgr-2)
gcagccatccctgtccccaagctttcttgtaagaacctctgccac AAIPVPKLSCKNLCHRHQLF
cgtcaccagctattcattaacttccgggacctgggttggcacaag INFRDLGWHKWIIAPKGFM
tggatcattgcccccaaggggttcatggcaaattactgccatgg ANYCHGECPFSLTISLNSSN
agagtgtcccttctcactgaccatctctctcaacagctccaattat YAFMQALMHAVDPEIPQA
gctttcatgcaagccctgatgcatgccgttgacccagagatccc VCIPTKLSPISMLYQDNNDN
ccaggctgtgtgtatccccaccaagctgtctcccatttccatgctc VILRHYEDMVVDECGCG
taccaggacaataatgacaatgtcattctacgacattatgaagac (SEQ ID NO: 63)
atggtagtcgatgaatgtgggtgtggg (SEQ ID NO: 62) GDF-5(BMP-1
gccccactggccactcgccagggcaagcgacccagcaagaa APLATRQGKRPSKNLKARC 4)
ccttaaggctcgctgcagtcggaaggcactgcatgtcaacttca SRKALHVNFKDMGWDDWI
aggacatgggctgggacgactggatcatcgcaccccttgagta IAPLEYEAFHCEGLCEFPLR
cgaggctttccactgcgaggggctgtgcgagttcccattgcgct SHLEPTNHAVIQTLMNSMD
cccacctggagcccacgaatcatgcagtcatccagaccctgat PESTPPTCCVPTRLSPISILFI
gaactccatggaccccgagtccacaccacccacctgctgtgtg DSANNVVYKQYEDMVVES
cccacgcggctgagtcccatcagcatcctcttcattgactctgcc CGCR
aacaacgtggtgtataagcagtatgaggacat (SEQ ID NO: 65)
ggtcgtggagtcgtgtggctgcagg (SEQ ID NO: 64) GDF-6(BMP-1
acggccttcgccagtcgccatggcaagcggcacggcaagaag TAFASRHGKRHGKKSRLRC 3)
tccaggctacgctgcagcaagaagcccctgcacgtgaacttca SKKPLHVNFKELGWDDWII
aggagctgggctgggacgactggattatcgcgcccctggagta APLEYEAYHCEGVCDFPLR
cgaggcctatcactgcgagggtgtatgcgacttcccgctgcgct SHLEPTNHAIIQTLMNSMDP
cgcacctggagcccaccaaccacgccatcatccagacgctgat GSTPPSCCVPTKLTPISILYID
gaactccatggaccccggctccaccccgcccagctgctgcgtg AGNNVVYKQYEDMVVESC
cccaccaaattgactcccatcagcattctatacatcgacgcggg GCR(SEQ ID NO: 67)
caataatgtggtctacaagcagtacgaggacatggtggtggagt cgtgcggctgcagg (SEQ ID
NO: 66) GDF-7(BMP-1 acggcgttggccgggacgcggacatcgcagggcagcggcg
TALAGTRTSQGSGGGAGRG 2) ggggcgcgggccggggccacgggcgcaggggccggagcc
HGRRGRSRCSRKPLHVDFK gctgcagccgcaagccgttgcacgtggacttcaaggagctcgg
ELGWDDWIIAPLDYEAYHC ctgggacgactggatcatcgcgccgctggactacgaggcgtac
EGLCDFPLRSHLEPTNHAII cactgcgagggcctttgcgacttccctttgcgttcgcacctcgag
QTLLNSMAPDAAPASCCVP cccaccaaccatgccatcattcagacgctgctcaactccatggc
ARLSPISILYIDAANNVVYK accagacgcggcgccggcctcctgctgtgtgccagcgcgcct
QYEDMVVEACGCR cagccccatcagcatcctctacatcgacgccgccaacaacgttg (SEQ ID
NO: 69) tctacaagcaatacgaggacatggtggtggaggcctgcggctg cagg (SEQ ID
NO: 68) GDF-8(Myosta-
gattttggtcttgactgtgatgagcactcaacagaatcacgatgct DFGLDCDEHSTESRCCRYP
tin) gtcgttaccctctaactgtggattttgaagcttttggatgggattgg
LTVDFEAFGWDWIIAPKRY attatcgctcctaaaagatataaggccaattactgctctggagagt
KANYCSGECEFVFLQKYPH
gtgaatttgtatttttacaaaaatatcctcatactcatctggtacacc
THLVHQANPRGSAGPCCTP aagcaaaccccagaggttcagcaggcccttgctgtactcccac
TKMSPINMLYFNGKEQIIYG aaagatgtctccaattaatatgctatattttaatggcaaagaacaa
KIPAMVVDRCGCS ataatatatgggaaaattccagcgatggtagtagaccgctgtgg (SEQ ID
NO: 71) gtgctca (SEQ ID NO: 70) GDF-9
ggtcaggaaactgtcagttctgaattgaagaagcccttgggccc GQETVSSELKKPLGPASFNL
agcttccttcaatctgagtgaatacttcagacaatttcttcttcccca
SEYFRQFLLPQNECELHDFR aaatgagtgtgagctccatgactttagacttagctttagtcagctg
LSFSQLKWDNWIVAPHRYN aagtgggacaactggattgtggctccgcacaggtacaaccctc
PRYCKGDCPRAVGHRYGSP gatactgtaaaggggactgtccaagggcagttggacatcggtat
VHTMVQNIIYEKLDSSVPRP ggctctccagttcacaccatggtacagaacatcatctatgagaa
SCVPAKYSPLSVLT1EPDGSI gctggactcctcagtgccaagaccgtcatgtgtacctgccaaat
AYKEYEDMIATKCTCR acagccccttgagtgttttgaccattgagcccgatggctcaattg (SEQ
ID NO: 73) cctataaagagtacgaagatatgatagctacaaagtgcacctgt cgt(SEQ ID
NO: 72) GDF-10(BMP- aagacgatgcagaaagcccggaggaagcagtgggatgagcc
KTMQKARRKQWDEPRVCS 3b) gagggtgtgctcccggaggtacctgaaggtggacttcgcagac
RRYLKVDFADIGWNEWIISP atcggctggaatgaatggataatctcaccgaaatcttttgatgcct
KSFDAYYCAGACEFPMPKI actactgcgcgggagcatgtgagttccccatgcctaagatcgtt
VRPSNHATIQSIVRAVGIIPG cgtccatccaaccatgccaccatccagagcattgtcagggctgt
IPEPCCVPDKMNSLGVLFLD gggcatcatccctggcatcccagagccctgctgtgttcccgata
ENRNVVLKVYPNMSVDTC agatgaactcccttggggtcctcttcctggatgagaat30lcgg ACR
aatgtggttctgaaggtgtaccccaacatgtccgtggacacctgt (SEQ ID NO: 75)
gcctgccggtga (SEQ ID NO: 74) GDF-11(BMP-
aacctgggtctggactgcgacgagcactcaagcgagtcccgct NLGLDCDEHSSESRCCRYP 11)
gctgccgatatcccctcacagtggactttgaggctttcggctggg LTVDFEAFGWDWIIAPKRY
actggatcatcgcacctaagcgctacaaggccaactactgctcc KANYCSGQCEYMFMQKYP
ggccagtgcgagtacatgttcatgcaaaaatatccgcataccca HTHLVQQANPRGSAGPCCT
tttggtgcagcaggccaatccaagaggctctgctgggccctgtt PTKMSPINMLYFNDKQQIIY
gtacccccaccaagatgtccccaatcaacatgctctacttcaatg GKIPGMVVDRCGCS
acaagcagcagattatctacggcaagatccctggcatggtggtg (SEQ ID NO: 77)
gatcgctgtggctgctct (SEQ ID NO: 76) GDF-15
gcgcgcaacggggaccactgtccgctcgggcccgggcgttgc ARNGDDCPLGPGRCCRLHT
tgccgtctgcacacggtccgcgcgtcgctggaagacctgggct VRASLEDLGWADWVLSPR
gggccgattgggtgctgtcgccacgggaggtgcaagtgaccat EVQVTMCIGACPSQFRAAN
gtgcatcggcgcgtgcccgagccagttccgggcggcaaacat MHAQIKTSLHRLKPDTEPA
gcacgcgcagatcaagacgagcctgcaccgcctgaagcccg PCCVPASYNPMVLIQKTDT
acacggtgccagcgccctgctgcgtgcccgccagctacaatcc GVSLQTYDDLLAKDCHCI
catggtgctcattcaaaagaccgacaccggggtgtcgctccag (SEQ ID NO: 79)
acctatgatgacttgttagccaaagactgccactgcata (SEQ ID NO: 78) Nodal
catcacttgccagacagaagtcaactgtgtcggaaggtcaagtt HHLPDRSQLCRKVKFQVDF
ccaggtggacttcaacctgatcggatggggctcctggatcatct NLIGWGSWIIYPKQYNAYR
accccaagcagtacaacgcctatcgctgtgagggcgagtgtcc CEGECPNPVGEEFHPTNHA
taatcctgttggggaggagtttcatccgaccaaccatgcatacat YIQSLLKRYQPHRVPSTCCA
ccagagtctgctgaaacgttaccagccccaccgagtcccttcca PVKTKPLSMLYVDNGRVLL
cttgttgtgccccagtgaagaccaagccgctgagcatgctgtat DHHKDMIVEECGCL
gtggataatggcagagtgctcctagatcaccataaagacatgat (SEQ ID NO: 81)
cgtggaagaatgtgggtgcctc (SEQ ID NO: 80) Activin-.beta.A
ggcctggagtgcgacggcaaggtcaacatctgctgtaagaaac GLECDGKVNICCKKQFFVS
agttctttgtcagtttcaaggacatcggctggaatgactggatcat FKDIGWNDWIIAPSGYHAN
tgctccctctggctatcatgccaactactgcgagggtgagtgcc YCEGECPSHIAGTSGSSLSF
cgagccatatagcaggcacgtccgggtcctcactgtccttccac HSTVINHYRMRGHSPFANL
tcaacagtcatcaaccactacgcatgcggccatagcccctttgc KSCCVPTKLRPMSMLYYDD
caacctcaaatcgtgctgtgtgcccaccaagctgagacccatgt GQNIIKKDIQNMiVEECGCS
ccatgttgtactatgatgatggtcaaaacatcatcaaaaaggaca (SEQ ID NO: 83)
ttcagaacatgatcgtggaggagtgcgggtgctcc (SEQ ID NO: 82) Activin-.beta.B
ggcctggagtgcgatggccggaccaacctctgttgcaggcaac GLECDGRTNLCCRQQFFIDF
agttcttcattgacttccgcctcatcggctggaacgactggatcat RLIGWNDWIIAPTGYYGNY
agcacccaccggctactacggcaactactgtgagggcagctgc CEGSCPAYLAGVPGSASSF
ccagcctacctggcaggggtccccggctctgcctcctccttcca HTAVVNQYRMRGLNPGTV
cacggctgtggtgaaccagtaccgcatgcggggtctgaacccc NSCCIPTKLSTMSMLYFDDE
ggcacggtgaactcctgctgcattcccaccaagctgagcaccat YNIVKRDVPNMIVEECGCA
gtccatgctgtacttcgatgatgagtacaacatcgtcaagcggg (SEQ ID NO: 85)
acgtgcccaacatgattgtggaggagtgcggctgcgcc (SEQ ID NO: 84)
Activin-.beta.C ggcatcgactgccaaggagggtccaggatgtgctgtcgacaa
GIDCQGGSRMCCRQFFFVD gagttttttgtggacttccgtgagattggctggcacgactggatca
FREIGWHDWIIQPEGYAMN tccagcctgagggctacgccatgaacttctgcatagggcagtgc
FCIGQCPLHIAGMPGIAASF ccactacacatagcaggcatgcctggtattgctgcctcctttcac
HTAVLNLLKANTAAGTTG
actgcagtgctcaatcttctcaaggccaacacagctgcaggcac GGSCCVPTARRPLSLLYYD
cactggagggggctcatgctgtgtacccacggcccggcgccc RDSNIVKTDIPDMVVEACG
cctgtctctgctctattatgacagggacagcaacattgtcaagact CS(SEQ ID NO: 87)
gacatacctgacatggtagtagaggcctgtgggtgcagt (SEQ ID NO: 86)
Activin-.beta.E acccccacctgtgagcctgcgacccccttatgttgcaggcgag
TPTCEPATPLCCRRDHYVD accattacgtagacttccaggaactgggatggcgggactggat
FQELGWRDWILQPEGYQLN actgcagcccgaggggtaccagctgaattactgcagtgggcag
YCSGQCPPHLAGSPGIAASF tgccctccccacctggctggcagcccaggcattgctgcctctttc
HSAVFSLLKANNPWPASTS cattctgccgtcttcagcctcctcaaagccaacaatccttggcct
CCVPTARRPLSLLYLDHNG gccagtacctcctgttgtgtccctactgcccgaaggcccctctct
NVVKTDVPDMVVEACGCS ctcctctacctggatcataatggcaatgtggtcaagacggatgtg
(SEQ ID NO: 89) ccagatatggtggtggaggcctgtggctgcagc (SEQ ID NO: 88)
Inhibin-.alpha. tcaactcccctgatgtcctggccttggtctccctctgctctgcgcc
STPLMSWPWSPSALRLLQR tgctgcagaggcctccggaggaaccggctgcccatgccaact
PPEEPAAHANCHRVALNISF gccacagagtagcactgaacatctccttccaggagctgggctg
QELGWERWIVYPPSFIFHYC ggaacggtggatcgtgtaccctcccagtttcatcttccactactgt
HGGCGLHIPPNLSLPVPGAP catggtggttgtgggctgcacatcccaccaaacctgtcccttcca
PTPAQPYSLLPGAQPCCAAL gtccctggggctccccctaccccagcccagccctactccttgct
PGTMRPLHVRTTSDGGYSF gccaggggcccagccctgctgtgctgctctcccagggaccatg
KYETVPNLLTQHCACI aggcccctacatgtccgcaccacctcggatggaggttactctttc (SEQ
ID NO: 91) aagtatgagacagtgcccaaccttctcacgcagcactgtgcttgt atc (SEQ
ID NO: 90) TGF-.beta.1 gccctggacaccaactattgcttcagctccacggagaagaactg
ALDTNYCFSSTEKNCCVRQ ctgcgtgcggcagctgtacattgacttccgcaaggacctcggct
LYIDFRKDLGWKWIHEPKG ggaagtggatccacgagcccaagggctaccatgccaacttctg
YHANFCLGPCPYIWSLDTQ cctcgggccctgcccctacatttggagcctggacacgcagtac
YSKVLALYNQHNPGASAAP agcaaggtcctggccctgtacaaccagcataacccgggcgcct
CCVPQALEPLPIVYYVGRKP cggcggcgccgtgctgcgtgccgcaggcgctggagccgctg
KVEQLSNMIVRSCKCS cccatcgtgtactacgtgggccgcaagcccaaggtggagcag (SEQ ID
NO: 93) ctgtccaacatgatcgtgcgctcctgcaagtgcagc (SEQ ID NO: 92
TGF-.beta.2 gctttggatgcggcctattgctttagaaatgtgcaggataattgct
ALDAAYCFRNVQDNCCLRP gcctacgtccactttacattgatttcaagagggatctagggtgga
LYIDFKRDLGWKWIHEPKG aatggatacacgaacccaaagggtacaatgccaacttctgtgct
YNANFCAGACPYLWSSDT ggagcatgcccgtatttatggagttcagacactcagcacagcag
QHSRVLSLYNTINPEASASP ggtcctgagcttatataataccataaatccagaagcatctgcttct
CCVSQDLEPLTILYYIGKTP
ccttgctgcgtgtcccaagatttagaacctctaaccattctctacta KIEQLSNMIVKSCKCS
cattggcaaaacacccaagattgaacagctttctaatatgattgta (SEQ ID NO: 95)
aagtcttgcaaatgcagc (SEQ ID NO: 94) TGF-.beta.3
gctttggacaccaattactgcttccgcaacttggaggagaactgc ALDTNYCFRNLEENCCVRP
tgtgtgcgccccctctacattgacttccgacaggatctgggctgg LYIDFRQDLGWKWVHEPK
aagtgggtccatgaacctaagggctactatgccaacttctgctca GYYANFCSGPCPYLRSADT
ggcccttgcccatacctccgcagtgcagacacaacccacagca THSTVLGLYNTLNPEASASP
cggtgctgggactgtacaacactctgaaccctgaagcatctgcc CCVPQDLEPLTILYYVGRTP
tcgccttgctgcgtgccccaggacctggagcccctgaccatcct KVEQLSNMVVKSCKCS
gtactatgttgggaggacccccaaagtggagcagctctccaac (SEQ ID NO: 97)
atggtggtgaagtcttgtaaatgtagc (SEQ ID NO: 96)
Sequence CWU 1
1
1501345DNAArtificial SequenceBMP-2 polynucleotide coding sequence
1atg caa gcc aaa cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48
Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10
15 aga cac cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg
96Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp
20 25 30 att gtg gct ccc ccg ggg tat cac gcc ttt tac tgc cac gga
gaa tgc 144Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly
Glu Cys 35 40 45 cct ttt cct ctg gct gat cat ctg aac tcc act aat
cat gcc att gtt 192Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn
His Ala Ile Val 50 55 60 cag acg ttg gtc aac tct gtt aac tct aag
att cct aag gca tgc tgt 240Gln Thr Leu Val Asn Ser Val Asn Ser Lys
Ile Pro Lys Ala Cys Cys 65 70 75 gtc ccg aca gaa ctc agt gct atc
tcg atg ctg tac ctt gac gag aat 288Val Pro Thr Glu Leu Ser Ala Ile
Ser Met Leu Tyr Leu Asp Glu Asn 80 85 90 95 gaa aag gtt gta tta aag
aac tat cag gac atg gtt gtg gag ggt tgt 336Glu Lys Val Val Leu Lys
Asn Tyr Gln Asp Met Val Val Glu Gly Cys 100 105 110 ggg tgt cgc
345Gly Cys Arg 2114PRTArtificial SequenceSynthetic Construct 2Gln
Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg 1 5 10
15 His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp Ile
20 25 30 Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu
Cys Pro 35 40 45 Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His
Ala Ile Val Gln 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile
Pro Lys Ala Cys Cys Val 65 70 75 80 Pro Thr Glu Leu Ser Ala Ile Ser
Met Leu Tyr Leu Asp Glu Asn Glu 85 90 95 Lys Val Val Leu Lys Asn
Tyr Gln Asp Met Val Val Glu Gly Cys Gly 100 105 110 Cys Arg
31281DNAHomo SapiensCDS(1)..(1281) 3atg ccc ttg ctt tgg ctg aga gga
ttt ctg ttg gca agt tgc tgg att 48Met Pro Leu Leu Trp Leu Arg Gly
Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 ata gtg agg agt tcc ccc
acc cca gga tcc gag ggg cac agc gcg gcc 96Ile Val Arg Ser Ser Pro
Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 ccc gac tgt ccg
tcc tgt gcg ctg gcc gcc ctc cca aag gat gta ccc 144Pro Asp Cys Pro
Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45 aac tct
cag cca gag atg gtg gag gcc gtc aag aag cac att tta aac 192Asn Ser
Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50 55 60
atg ctg cac ttg aag aag aga ccc gat gtc acc cag ccg gta ccc aag
240Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro Lys
65 70 75 80 gcg gcg ctt ctg aac gcg atc aga aag ctt cat gtg ggc aaa
gtc ggg 288Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly Lys
Val Gly 85 90 95 gag aac ggg tat gtg gag ata gag gat gac att gga
agg agg gca gaa 336Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile Gly
Arg Arg Ala Glu 100 105 110 atg aat gaa ctt atg gag cag acc tcg gag
atc atc acg ttt gcc gag 384Met Asn Glu Leu Met Glu Gln Thr Ser Glu
Ile Ile Thr Phe Ala Glu 115 120 125 tca gga aca gcc agg aag acg ctg
cac ttc gag att tcc aag gaa ggc 432Ser Gly Thr Ala Arg Lys Thr Leu
His Phe Glu Ile Ser Lys Glu Gly 130 135 140 agt gac ctg tca gtg gtg
gag cgt gca gaa gtc tgg ctc ttc cta aaa 480Ser Asp Leu Ser Val Val
Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 gtc ccc aag
gcc aac agg acc agg acc aaa gtc acc atc cgc ctc ttc 528Val Pro Lys
Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175 cag
cag cag aag cac ccg cag ggc agc ttg gac aca ggg gaa gag gcc 576Gln
Gln Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala 180 185
190 gag gaa gtg ggc tta aag ggg gag agg agt gaa ctg ttg ctc tct gaa
624Glu Glu Val Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser Glu
195 200 205 aaa gta gta gac gct cgg aag agc acc tgg cat gtc ttc cct
gtc tcc 672Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe Pro
Val Ser 210 215 220 agc agc atc cag cgg ttg ctg gac cag ggc aag agc
tcc ctg gac gtt 720Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys Ser
Ser Leu Asp Val 225 230 235 240 cgg att gcc tgt gag cag tgc cag gag
agt ggc gcc agc ttg gtt ctc 768Arg Ile Ala Cys Glu Gln Cys Gln Glu
Ser Gly Ala Ser Leu Val Leu 245 250 255 ctg ggc aag aag aag aag aaa
gaa gag gag ggg gaa ggg aaa aag aag 816Leu Gly Lys Lys Lys Lys Lys
Glu Glu Glu Gly Glu Gly Lys Lys Lys 260 265 270 ggc gga ggt gaa ggt
ggg gca gga gca gat gag gaa aag gag cag tcg 864Gly Gly Gly Glu Gly
Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser 275 280 285 cac aga cct
ttc ctc atg ctg cag gcc cgg cag tct gaa gac cac cct 912His Arg Pro
Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro 290 295 300 cat
cgc cgg cgt cgg cgg ggc ttg gag tgt gat ggc aag gtc aac atc 960His
Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile 305 310
315 320 tgc tgt aag aaa cag ttc ttt gtc agt ttc aag gac atc ggc tgg
aat 1008Cys Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp
Asn 325 330 335 gac tgg atc att gct ccc tct ggc tat cat gcc aac tac
tgc gag ggt 1056Asp Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr
Cys Glu Gly 340 345 350 gag tgc ccg agc cat ata gca ggc acg tcc ggg
tcc tca ctg tcc ttc 1104Glu Cys Pro Ser His Ile Ala Gly Thr Ser Gly
Ser Ser Leu Ser Phe 355 360 365 cac tca aca gtc atc aac cac tac cgc
atg cgg ggc cat agc ccc ttt 1152His Ser Thr Val Ile Asn His Tyr Arg
Met Arg Gly His Ser Pro Phe 370 375 380 gcc aac ctc aaa tcg tgc tgt
gtg ccc acc aag ctg aga ccc atg tcc 1200Ala Asn Leu Lys Ser Cys Cys
Val Pro Thr Lys Leu Arg Pro Met Ser 385 390 395 400 atg ttg tac tat
gat gat ggt caa aac atc atc aaa aag gac att cag 1248Met Leu Tyr Tyr
Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln 405 410 415 aac atg
atc gtg gag gag tgt ggg tgc tca tag 1281Asn Met Ile Val Glu Glu Cys
Gly Cys Ser 420 425 4426PRTHomo Sapiens 4Met Pro Leu Leu Trp Leu
Arg Gly Phe Leu Leu Ala Ser Cys Trp Ile 1 5 10 15 Ile Val Arg Ser
Ser Pro Thr Pro Gly Ser Glu Gly His Ser Ala Ala 20 25 30 Pro Asp
Cys Pro Ser Cys Ala Leu Ala Ala Leu Pro Lys Asp Val Pro 35 40 45
Asn Ser Gln Pro Glu Met Val Glu Ala Val Lys Lys His Ile Leu Asn 50
55 60 Met Leu His Leu Lys Lys Arg Pro Asp Val Thr Gln Pro Val Pro
Lys 65 70 75 80 Ala Ala Leu Leu Asn Ala Ile Arg Lys Leu His Val Gly
Lys Val Gly 85 90 95 Glu Asn Gly Tyr Val Glu Ile Glu Asp Asp Ile
Gly Arg Arg Ala Glu 100 105 110 Met Asn Glu Leu Met Glu Gln Thr Ser
Glu Ile Ile Thr Phe Ala Glu 115 120 125 Ser Gly Thr Ala Arg Lys Thr
Leu His Phe Glu Ile Ser Lys Glu Gly 130 135 140 Ser Asp Leu Ser Val
Val Glu Arg Ala Glu Val Trp Leu Phe Leu Lys 145 150 155 160 Val Pro
Lys Ala Asn Arg Thr Arg Thr Lys Val Thr Ile Arg Leu Phe 165 170 175
Gln Gln Gln Lys His Pro Gln Gly Ser Leu Asp Thr Gly Glu Glu Ala 180
185 190 Glu Glu Val Gly Leu Lys Gly Glu Arg Ser Glu Leu Leu Leu Ser
Glu 195 200 205 Lys Val Val Asp Ala Arg Lys Ser Thr Trp His Val Phe
Pro Val Ser 210 215 220 Ser Ser Ile Gln Arg Leu Leu Asp Gln Gly Lys
Ser Ser Leu Asp Val 225 230 235 240 Arg Ile Ala Cys Glu Gln Cys Gln
Glu Ser Gly Ala Ser Leu Val Leu 245 250 255 Leu Gly Lys Lys Lys Lys
Lys Glu Glu Glu Gly Glu Gly Lys Lys Lys 260 265 270 Gly Gly Gly Glu
Gly Gly Ala Gly Ala Asp Glu Glu Lys Glu Gln Ser 275 280 285 His Arg
Pro Phe Leu Met Leu Gln Ala Arg Gln Ser Glu Asp His Pro 290 295 300
His Arg Arg Arg Arg Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile 305
310 315 320 Cys Cys Lys Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly
Trp Asn 325 330 335 Asp Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn
Tyr Cys Glu Gly 340 345 350 Glu Cys Pro Ser His Ile Ala Gly Thr Ser
Gly Ser Ser Leu Ser Phe 355 360 365 His Ser Thr Val Ile Asn His Tyr
Arg Met Arg Gly His Ser Pro Phe 370 375 380 Ala Asn Leu Lys Ser Cys
Cys Val Pro Thr Lys Leu Arg Pro Met Ser 385 390 395 400 Met Leu Tyr
Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln 405 410 415 Asn
Met Ile Val Glu Glu Cys Gly Cys Ser 420 425 5116PRTArtificial
SequenceMature activin polypeptide homo sapiens 5Gly Leu Glu Cys
Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe 1 5 10 15 Phe Val
Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro 20 25 30
Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile 35
40 45 Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile
Asn 50 55 60 His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu
Lys Ser Cys 65 70 75 80 Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met
Leu Tyr Tyr Asp Asp 85 90 95 Gly Gln Asn Ile Ile Lys Lys Asp Ile
Gln Asn Met Ile Val Glu Glu 100 105 110 Cys Gly Cys Ser 115
6345DNAArtificial SequenceChimeric polypeptide 6atg caa gcc aaa cac
aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys His
Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac cct
ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg 96Arg His Pro
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 att
gtg gct ccc ccg ggg tat cac gcc ttt tac tgc cac gga gaa tgc 144Ile
Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35 40
45 cct ttt cct ctg gct gat cat ctg aac tcc act aat cat gcc att gtt
192Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val
50 55 60 cag acg ttg gtc aac tct gtt aac tct aag att cct aag gca
tgc tgt 240Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala
Cys Cys 65 70 75 80 gtc ccg aca gaa ctc agt gct atc tcg atg ttg tac
tat gat gat ggt 288Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr
Tyr Asp Asp Gly 85 90 95 cga aac atc atc aaa aag gac att cag aac
atg atc gtg gag gag tgt 336Arg Asn Ile Ile Lys Lys Asp Ile Gln Asn
Met Ile Val Glu Glu Cys 100 105 110 ggg tgc tca 345Gly Cys Ser 115
7115PRTArtificial SequenceSynthetic Construct 7Met Gln Ala Lys His
Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His Pro
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile
Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35 40
45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val
50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala
Cys Cys 65 70 75 80 Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr
Tyr Asp Asp Gly 85 90 95 Arg Asn Ile Ile Lys Lys Asp Ile Gln Asn
Met Ile Val Glu Glu Cys 100 105 110 Gly Cys Ser 115
8342DNAArtificial SequenceChimeric polypeptide 8atg caa gcc aaa cac
caa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys His
Gln Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac cct
ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg 96Arg His Pro
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 att
gtg gct ccc ccg ggg tat cac gcc ttt tac tgc cac gga gaa tgc 144Ile
Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35 40
45 cct ttt cct ctg gct gat cat ctg aac tcc act aat cat gcc att gtt
192Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val
50 55 60 cag acg ttg gtc aac tct gtt aac tct aag att cct aag gca
tgc tgt 240Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala
Cys Cys 65 70 75 80 gtc ccg acc aag ctg aga ccc tcc atg ttg tac tat
gat gat ggt caa 288Val Pro Thr Lys Leu Arg Pro Ser Met Leu Tyr Tyr
Asp Asp Gly Gln 85 90 95 aac atc atc aaa aag gac att cag aac atg
atc gtg gag gag tgt ggg 336Asn Ile Ile Lys Lys Asp Ile Gln Asn Met
Ile Val Glu Glu Cys Gly 100 105 110 tgc tca 342Cys Ser
9114PRTArtificial SequenceSynthetic Construct 9Met Gln Ala Lys His
Gln Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His Pro
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile
Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35 40
45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val
50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala
Cys Cys 65 70 75 80 Val Pro Thr Lys Leu Arg Pro Ser Met Leu Tyr Tyr
Asp Asp Gly Gln 85 90 95 Asn Ile Ile
Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu Cys Gly 100 105 110 Cys
Ser 10345DNAArtificial SequenceChimeric polypeptide 10atg caa gcc
aaa cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga
cac cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg 96Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 att gtg gct ccc ccg ggg tat cac gcc ttt tac tgc cac gga gaa tgc
144Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys
35 40 45 cct ttt cct ctg gct gat cat ctg aac tcc act aat cat gcc
att gtt 192Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala
Ile Val 50 55 60 cag acg ttg gtc aac tct gtt aac tct aag att cct
aag gca tgc tgt 240Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro
Lys Ala Cys Cys 65 70 75 80 gtc ccg acc aag ctg aga ccc atg tcc atg
ttg tac tat gat gag aat 288Val Pro Thr Lys Leu Arg Pro Met Ser Met
Leu Tyr Tyr Asp Glu Asn 85 90 95 gaa aag gtt gta tta aag aac tat
cag gac atg gtt gtg gag ggt tgt 336Glu Lys Val Val Leu Lys Asn Tyr
Gln Asp Met Val Val Glu Gly Cys 100 105 110 ggg tgt cgc 345Gly Cys
Arg 115 11115PRTArtificial SequenceSynthetic Construct 11Met Gln
Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15
Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20
25 30 Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu
Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His
Ala Ile Val 50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile
Pro Lys Ala Cys Cys 65 70 75 80 Val Pro Thr Lys Leu Arg Pro Met Ser
Met Leu Tyr Tyr Asp Glu Asn 85 90 95 Glu Lys Val Val Leu Lys Asn
Tyr Gln Asp Met Val Val Glu Gly Cys 100 105 110 Gly Cys Arg 115
12360DNAArtificial SequenceChimeric polypeptide 12atg caa gcc aaa
cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac
cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg 96Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
att gtg gct ccc ccg ggg tat cac gcc ttt tac tgc cac gga gaa tgc
144Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys
35 40 45 cct tct cat ata gca ggc acg tcc ggg tcc tca ctg tcc ttc
cac tca 192Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 acg ttg gtc aac cac tac cgc atg cgg ggc cat agc
ccc ttt gcc aac 240Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser
Pro Phe Ala Asn 65 70 75 80 ctc aaa tcg tgc tgt gtc ccg acc aag ctg
aga ccc atg tcc atg ttg 288Leu Lys Ser Cys Cys Val Pro Thr Lys Leu
Arg Pro Met Ser Met Leu 85 90 95 tac tat gat gat ggt caa aac atc
atc aaa aag gac att cag aac atg 336Tyr Tyr Asp Asp Gly Gln Asn Ile
Ile Lys Lys Asp Ile Gln Asn Met 100 105 110 atc gtg gag gag tgt ggg
tgc tca 360Ile Val Glu Glu Cys Gly Cys Ser 115 120
13120PRTArtificial SequenceSynthetic Construct 13Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35
40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His
Ser 50 55 60 Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser Pro
Phe Ala Asn 65 70 75 80 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg
Pro Met Ser Met Leu 85 90 95 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile
Lys Lys Asp Ile Gln Asn Met 100 105 110 Ile Val Glu Glu Cys Gly Cys
Ser 115 120 14360DNAArtificial SequenceChimeric polypeptide 14atg
caa gcc aaa cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met
Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10
15 aga cac cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg
96Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp
20 25 30 att gtg gct ccc ccg ggg tat cac gcc ttt tac tgc cac gga
gaa tgc 144Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly
Glu Cys 35 40 45 cct tct cat ata gca ggc acg tcc ggg tcc tca ctg
tcc tta cac tca 192Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu
Ser Leu His Ser 50 55 60 acg ttg gtc aac cac tac cgc atg cgg ggc
cat agc ccc ttt gcc aac 240Thr Leu Val Asn His Tyr Arg Met Arg Gly
His Ser Pro Phe Ala Asn 65 70 75 80 ctc aaa tcg tgc tgt gtc ccg aca
gag ctc agt gct atc tcg atg ttg 288Leu Lys Ser Cys Cys Val Pro Thr
Glu Leu Ser Ala Ile Ser Met Leu 85 90 95 tac tat gat gat ggt caa
aac atc atc aaa aag gac att cag aac atg 336Tyr Tyr Asp Asp Gly Gln
Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 100 105 110 atc gtg gag gag
tgt ggg tgc tca 360Ile Val Glu Glu Cys Gly Cys Ser 115 120
15120PRTArtificial SequenceSynthetic Construct 15Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35
40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Leu His
Ser 50 55 60 Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser Pro
Phe Ala Asn 65 70 75 80 Leu Lys Ser Cys Cys Val Pro Thr Glu Leu Ser
Ala Ile Ser Met Leu 85 90 95 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile
Lys Lys Asp Ile Gln Asn Met 100 105 110 Ile Val Glu Glu Cys Gly Cys
Ser 115 120 16360DNAArtificial SequenceChimeric polypeptide 16atg
caa gcc aaa cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met
Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10
15 aga cac cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg
96Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp
20 25 30 atc att gct ccc tct ggc tat cat gcc aac tac tgc gag gga
gaa tgc 144Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly
Glu Cys 35 40 45 cct tcc cat ata gca ggc acg tcc ggg tcc tca ctg
tcc ttc cat tca 192Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu
Ser Phe His Ser 50 55 60 acg ttg gtc aac cac tac cgc atg cgg ggc
cat agc ccc ttt gcc aac 240Thr Leu Val Asn His Tyr Arg Met Arg Gly
His Ser Pro Phe Ala Asn 65 70 75 80 ctc aaa tcg tgc tgt gtc ccg acc
aag ctg aga ccc atg tcc atg ttg 288Leu Lys Ser Cys Cys Val Pro Thr
Lys Leu Arg Pro Met Ser Met Leu 85 90 95 tac tat gat gat ggt caa
aac atc atc aaa aag gac att cag aac atg 336Tyr Tyr Asp Asp Gly Gln
Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 100 105 110 atc gtg gag gag
tgt ggg tgc tca 360Ile Val Glu Glu Cys Gly Cys Ser 115 120
17120PRTArtificial SequenceSynthetic Construct 17Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 35
40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His
Ser 50 55 60 Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser Pro
Phe Ala Asn 65 70 75 80 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg
Pro Met Ser Met Leu 85 90 95 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile
Lys Lys Asp Ile Gln Asn Met 100 105 110 Ile Val Glu Glu Cys Gly Cys
Ser 115 120 18360DNAArtificial SequenceChimeric polypeptide 18atg
caa gcc aaa cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met
Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10
15 aga cac cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg
96Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp
20 25 30 atc att gct ccc tct ggc tat cat gcc aac tac tgc gag gga
gaa tgc 144Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly
Glu Cys 35 40 45 cct tcc cat ata gca ggc acg tcc ggg tcc tca ctg
tcc ttc cat tca 192Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu
Ser Phe His Ser 50 55 60 acg gtg atc aac cac tac cgc atg cgg ggc
cat agc ccc ttt gcc aac 240Thr Val Ile Asn His Tyr Arg Met Arg Gly
His Ser Pro Phe Ala Asn 65 70 75 80 ctc aaa tcg tgc tgt gtc ccg acc
aag ctg aga ccc atg tcc atg ttg 288Leu Lys Ser Cys Cys Val Pro Thr
Lys Leu Arg Pro Met Ser Met Leu 85 90 95 tac tat gat gat ggt caa
aac atc atc aaa aag gac att cag aac atg 336Tyr Tyr Asp Asp Gly Gln
Asn Ile Ile Lys Lys Asp Ile Gln Asn Met 100 105 110 atc gtg gag gag
tgt ggg tgc tca 360Ile Val Glu Glu Cys Gly Cys Ser 115 120
19120PRTArtificial SequenceSynthetic Construct 19Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 35
40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His
Ser 50 55 60 Thr Val Ile Asn His Tyr Arg Met Arg Gly His Ser Pro
Phe Ala Asn 65 70 75 80 Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg
Pro Met Ser Met Leu 85 90 95 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile
Lys Lys Asp Ile Gln Asn Met 100 105 110 Ile Val Glu Glu Cys Gly Cys
Ser 115 120 20357DNAArtificial SequenceChimeric polypeptide 20atg
caa gcc aaa cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met
Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10
15 aaa cag ttc ttt gtc agt ttc aag gac atc ggg tgg aat gac tgg atc
96Lys Gln Phe Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile
20 25 30 att gct ccc tct ggc tat cat gcc aac tac tgc gag gga gaa
tgc cct 144Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu
Cys Pro 35 40 45 tcc cat ata gca ggc acg tcc ggg tcc tca ctg tcc
ttc cat tca acg 192Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser
Phe His Ser Thr 50 55 60 ttg gtc aac cac tac cgc atg cgg ggc cat
agc ccc ttt gcc aac ctc 240Leu Val Asn His Tyr Arg Met Arg Gly His
Ser Pro Phe Ala Asn Leu 65 70 75 80 aaa tcg tgc tgt gtc ccg acc aag
ctg aga ccc atg tcc atg ttg tac 288Lys Ser Cys Cys Val Pro Thr Lys
Leu Arg Pro Met Ser Met Leu Tyr 85 90 95 tat gat gat ggt caa aac
atc atc aaa aag gac att cag aac atg atc 336Tyr Asp Asp Gly Gln Asn
Ile Ile Lys Lys Asp Ile Gln Asn Met Ile 100 105 110 gtg gag gag tgt
ggg tgc tca 357Val Glu Glu Cys Gly Cys Ser 115 21119PRTArtificial
SequenceSynthetic Construct 21Met Gln Ala Lys His Lys Gln Arg Lys
Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Lys Gln Phe Phe Val Ser Phe
Lys Asp Ile Gly Trp Asn Asp Trp Ile 20 25 30 Ile Ala Pro Ser Gly
Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro 35 40 45 Ser His Ile
Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr 50 55 60 Leu
Val Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu 65 70
75 80 Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu
Tyr 85 90 95 Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln
Asn Met Ile 100 105 110 Val Glu Glu Cys Gly Cys Ser 115
22360DNAArtificial SequenceChimeric polypeptide 22atg caa gcc aaa
cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac
cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg 96Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
atc att gct ccc tct ggc tat cat gcc aac tac tgc gag gga gaa tgc
144Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys
35 40 45 cct tct cat ata gca ggc acg tcc ggg tcc tca ctg tcc ttc
cac tca 192Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 acg ttg gtc aac cac tac cgc atg cgg ggc cat agc
ccc ttt gcc aac 240Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser
Pro Phe Ala Asn 65 70 75 80 ctc aaa tcg tgc tgt gtc ccg acc aag ctg
aga ccc atg tcc atg ttg 288Leu Lys Ser Cys Cys Val Pro Thr Lys Leu
Arg Pro Met Ser Met Leu 85 90 95 tac ctt gac gag aat gaa aag gtt
gta tta aag aac tat cag gac atg 336Tyr Leu Asp Glu Asn Glu Lys Val
Val Leu Lys Asn Tyr Gln
Asp Met 100 105 110 gtt gtg gag ggt tgt ggg tgt cgc 360Val Val Glu
Gly Cys Gly Cys Arg 115 120 23120PRTArtificial SequenceSynthetic
Construct 23Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser
Cys Lys 1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly
Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn
Tyr Cys Glu Gly Glu Cys 35 40 45 Pro Ser His Ile Ala Gly Thr Ser
Gly Ser Ser Leu Ser Phe His Ser 50 55 60 Thr Leu Val Asn His Tyr
Arg Met Arg Gly His Ser Pro Phe Ala Asn 65 70 75 80 Leu Lys Ser Cys
Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 85 90 95 Tyr Leu
Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met 100 105 110
Val Val Glu Gly Cys Gly Cys Arg 115 120 24360DNAArtificial
SequenceChimeric polypeptide 24atg caa gcc aaa cac aaa cag cgg aaa
cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys His Lys Gln Arg Lys
Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac cct ttg tac gtg gac
ttc agt gac gtg ggg tgg aat gac tgg 96Arg His Pro Leu Tyr Val Asp
Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 atc att gct ccc tct
ggc tat cat gcc aac tac tgc gag gga gaa tgc 144Ile Ile Ala Pro Ser
Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 35 40 45 cct tct cat
ata gca ggc acg tcc ggg tcc tca ctg tcc ttc cac tca 192Pro Ser His
Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 50 55 60 acg
ttg gtc aac cac tac cgc atg cgg ggc cat agc ccc ttt gcc aac 240Thr
Leu Val Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 65 70
75 80 ctc aaa tcg tgc tgt gtc ccg aca gaa ctc agt gct atc tcg atg
ttg 288Leu Lys Ser Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met
Leu 85 90 95 tac ctt gac gag aat gaa aag gtt gta tta aag aac tat
cag gac atg 336Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr
Gln Asp Met 100 105 110 gtt gtg gag ggt tgt ggg tgt cgc 360Val Val
Glu Gly Cys Gly Cys Arg 115 120 25120PRTArtificial
SequenceSynthetic Construct 25Met Gln Ala Lys His Lys Gln Arg Lys
Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His Pro Leu Tyr Val Asp
Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Ser
Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 35 40 45 Pro Ser His
Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 50 55 60 Thr
Leu Val Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn 65 70
75 80 Leu Lys Ser Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met
Leu 85 90 95 Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr
Gln Asp Met 100 105 110 Val Val Glu Gly Cys Gly Cys Arg 115 120
26360DNAArtificial SequenceChimeric polypeptide 26atg caa gcc aaa
cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac
cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg 96Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
atc att gct ccc tct ggc tat cat gcc aac tac tgc gag gga gaa tgc
144Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys
35 40 45 cct tct cat ata gca ggc acg tcc ggg tcc tca ctg tcc ttc
cac tca 192Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 acg ttg gtc aac cac tac cgc atg cgg ggc cat agc
ccc ttt gcc aac 240Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser
Pro Phe Ala Asn 65 70 75 80 ctc aaa tcg tgc tgt gtc ccg aca gaa ctc
agt gct atc tcg atg ttg 288Leu Lys Ser Cys Cys Val Pro Thr Glu Leu
Ser Ala Ile Ser Met Leu 85 90 95 tac tat gat gat ggt caa aac atc
atc aaa aag gac att cag aac atg 336Tyr Tyr Asp Asp Gly Gln Asn Ile
Ile Lys Lys Asp Ile Gln Asn Met 100 105 110 atc gtg gag gag tgt ggg
tgc tca 360Ile Val Glu Glu Cys Gly Cys Ser 115 120
27120PRTArtificial SequenceSynthetic Construct 27Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys 35
40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His
Ser 50 55 60 Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser Pro
Phe Ala Asn 65 70 75 80 Leu Lys Ser Cys Cys Val Pro Thr Glu Leu Ser
Ala Ile Ser Met Leu 85 90 95 Tyr Tyr Asp Asp Gly Gln Asn Ile Ile
Lys Lys Asp Ile Gln Asn Met 100 105 110 Ile Val Glu Glu Cys Gly Cys
Ser 115 120 28345DNAArtificial SequenceChimeric polypeptide 28atg
caa gcc aaa cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met
Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10
15 aga cac cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg
96Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp
20 25 30 atc att gct ccc tct ggc tat cat gcc aac tac tgc gac gga
gaa tgc 144Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Asp Gly
Glu Cys 35 40 45 cct ttt cct ctg gct gat cat ctg aac tcc act aat
cat gcc att gtt 192Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn
His Ala Ile Val 50 55 60 cag acg ttg gtc aac tct gtt aac tct aag
att cct aag gca tgc tgt 240Gln Thr Leu Val Asn Ser Val Asn Ser Lys
Ile Pro Lys Ala Cys Cys 65 70 75 80 gtc ccg aca gaa ctc agt gct atc
tcg atg ttg tac tat gat gat ggt 288Val Pro Thr Glu Leu Ser Ala Ile
Ser Met Leu Tyr Tyr Asp Asp Gly 85 90 95 caa aac atc atc aaa aag
gac att cag aac atg atc gtg gag gag tgt 336Gln Asn Ile Ile Lys Lys
Asp Ile Gln Asn Met Ile Val Glu Glu Cys 100 105 110 ggg tgc tca
345Gly Cys Ser 115 29115PRTArtificial SequenceSynthetic Construct
29Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1
5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp
Trp 20 25 30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Asp
Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr
Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser
Lys Ile Pro Lys Ala Cys Cys 65 70 75 80 Val Pro Thr Glu Leu Ser Ala
Ile Ser Met Leu Tyr Tyr Asp Asp Gly 85 90 95 Gln Asn Ile Ile Lys
Lys Asp Ile Gln Asn Met Ile Val Glu Glu Cys 100 105 110 Gly Cys Ser
115 30345DNAArtificial SequenceChimeric polypeptide 30atg caa gcc
aaa cac aaa cag cgg aag cgt ctt aag tcc agc tgc aaa 48Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 agg
cac cct ttg tat gtg gac ttc agt gat gtg ggg tgg aat gac tgg 96Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 atc att gct ccc tct ggc tat cat gcc aac tac tgc gac gga gaa tgc
144Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Asp Gly Glu Cys
35 40 45 cct ttt cct ctg gct gat cat ctg aac tcc act aat cat gcc
att gtt 192Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala
Ile Val 50 55 60 cag acg ttg gtc aac tct gtt aac tct aag att cct
aag gca tgc tgt 240Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro
Lys Ala Cys Cys 65 70 75 80 gtc ccg acc aag ctg aga ccc atg tcc atg
ttg tac tat gat gat ggt 288Val Pro Thr Lys Leu Arg Pro Met Ser Met
Leu Tyr Tyr Asp Asp Gly 85 90 95 caa aac atc atc aaa aag gac att
cag aac atg atc gtg gag gag tgt 336Gln Asn Ile Ile Lys Lys Asp Ile
Gln Asn Met Ile Val Glu Glu Cys 100 105 110 ggg tgc tca 345Gly Cys
Ser 115 31115PRTArtificial SequenceSynthetic Construct 31Met Gln
Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15
Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20
25 30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Asp Gly Glu
Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His
Ala Ile Val 50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile
Pro Lys Ala Cys Cys 65 70 75 80 Val Pro Thr Lys Leu Arg Pro Met Ser
Met Leu Tyr Tyr Asp Asp Gly 85 90 95 Gln Asn Ile Ile Lys Lys Asp
Ile Gln Asn Met Ile Val Glu Glu Cys 100 105 110 Gly Cys Ser 115
32345DNAArtificial SequenceChimeric polypeptide 32atg caa gcc aaa
cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac
cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg 96Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
atc att gct ccc tct ggc tat cat gcc aac tac tgc gac gga gaa tgc
144Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Asp Gly Glu Cys
35 40 45 cct ttt cct ctg gct gat cat ctg aac tcc act aat cat gcc
att gtt 192Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala
Ile Val 50 55 60 cag acg ttg gtc aac tct gtt aac tct aag att cct
aag gca tgc tgt 240Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro
Lys Ala Cys Cys 65 70 75 80 gtc ccg acc aag ctg aga ccc atg tcc atg
ttg tac ctt gac gag aat 288Val Pro Thr Lys Leu Arg Pro Met Ser Met
Leu Tyr Leu Asp Glu Asn 85 90 95 gaa aag gtt gta tta aag aac tat
cag gac atg gtt gtg gag ggt tgt 336Glu Lys Val Val Leu Lys Asn Tyr
Gln Asp Met Val Val Glu Gly Cys 100 105 110 ggg tgt cgc 345Gly Cys
Arg 115 33115PRTArtificial SequenceSynthetic Construct 33Met Gln
Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15
Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20
25 30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Asp Gly Glu
Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His
Ala Ile Val 50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile
Pro Lys Ala Cys Cys 65 70 75 80 Val Pro Thr Lys Leu Arg Pro Met Ser
Met Leu Tyr Leu Asp Glu Asn 85 90 95 Glu Lys Val Val Leu Lys Asn
Tyr Gln Asp Met Val Val Glu Gly Cys 100 105 110 Gly Cys Arg 115
34348DNAArtificial SequenceChimeric polypeptide 34atg caa gcc aaa
cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys
His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac
cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg 96Arg His
Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30
att atc gcg cct gaa ggc tac gcc gcc tac tac tgt gag ggg gag tgt
144Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys
35 40 45 gcc ttc cct ctg aac tcc tac atg aac gcc acc aac cac gcc
atc gtg 192Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala
Ile Val 50 55 60 cag acg ctg gtc cac ttc atc aac ccg gaa acg gtg
ccc aag ccc tgc 240Gln Thr Leu Val His Phe Ile Asn Pro Glu Thr Val
Pro Lys Pro Cys 65 70 75 80 tgt gcg ccc acg cag ctc aat gcc atc tcc
gtc ctc tac ttc gat gac 288Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser
Val Leu Tyr Phe Asp Asp 85 90 95 agc tcc aac gtc atc ctg aag aaa
tac aga aac atg gtg gtc cgg gcc 336Ser Ser Asn Val Ile Leu Lys Lys
Tyr Arg Asn Met Val Val Arg Ala 100 105 110 tgt ggc tgc cac 348Cys
Gly Cys His 115 35116PRTArtificial SequenceSynthetic Construct
35Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1
5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp
Trp 20 25 30 Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu
Gly Glu Cys 35 40 45 Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr
Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val His Phe Ile Asn Pro
Glu Thr Val Pro Lys Pro Cys 65 70 75 80 Cys Ala Pro Thr Gln Leu Asn
Ala Ile Ser Val Leu Tyr Phe Asp Asp 85 90 95 Ser Ser Asn Val Ile
Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala 100 105 110 Cys Gly Cys
His 115 36351DNAArtificial SequenceChimeric polypeptide 36atg caa
gcc aaa cac aaa cag cgg aaa cgc ctt aag tcc agc tgt aag 48Met Gln
Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15
aga cac cct ttg tac gtg gac ttc agt gac gtg ggg tgg aat gac tgg
96Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp
Trp
20 25 30 att att gcc cca aaa gag tac gag gca tac gag tgt aag ggc
ggc tgt 144Ile Ile Ala Pro Lys Glu Tyr Glu Ala Tyr Glu Cys Lys Gly
Gly Cys 35 40 45 ttc ttt ccg ctg gcc gac gat gtc acc ccg acc aag
cac gca att gtc 192Phe Phe Pro Leu Ala Asp Asp Val Thr Pro Thr Lys
His Ala Ile Val 50 55 60 caa acc tta gtg cac ctg aag ttc cca acg
aaa gtg ggt aag gca tgt 240Gln Thr Leu Val His Leu Lys Phe Pro Thr
Lys Val Gly Lys Ala Cys 65 70 75 80 tgt gtg cca acc aag tta tct cca
att agc gtg ctg tat aag gat gat 288Cys Val Pro Thr Lys Leu Ser Pro
Ile Ser Val Leu Tyr Lys Asp Asp 85 90 95 atg ggc gtg ccg acg tta
aag tat cat tac gag ggc atg agc gtc gca 336Met Gly Val Pro Thr Leu
Lys Tyr His Tyr Glu Gly Met Ser Val Ala 100 105 110 gag tgt ggc tgc
cgc 351Glu Cys Gly Cys Arg 115 37117PRTArtificial SequenceSynthetic
Construct 37Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser
Cys Lys 1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly
Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Lys Glu Tyr Glu Ala Tyr
Glu Cys Lys Gly Gly Cys 35 40 45 Phe Phe Pro Leu Ala Asp Asp Val
Thr Pro Thr Lys His Ala Ile Val 50 55 60 Gln Thr Leu Val His Leu
Lys Phe Pro Thr Lys Val Gly Lys Ala Cys 65 70 75 80 Cys Val Pro Thr
Lys Leu Ser Pro Ile Ser Val Leu Tyr Lys Asp Asp 85 90 95 Met Gly
Val Pro Thr Leu Lys Tyr His Tyr Glu Gly Met Ser Val Ala 100 105 110
Glu Cys Gly Cys Arg 115 38348DNAArtificial SequenceChimeric
polypeptide 38atg caa gcc aaa cac aaa cag cgg aaa cgc ctt aag tcc
agc tgt aag 48Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser
Ser Cys Lys 1 5 10 15 aga cac cct ttg tac gtg gac ttc agt gac gtg
ggg tgg aat gac tgg 96Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val
Gly Trp Asn Asp Trp 20 25 30 att atc gcg ccg ctg gac tac gag gcg
tac cac tgc gag ggc cta tgc 144Ile Ile Ala Pro Leu Asp Tyr Glu Ala
Tyr His Cys Glu Gly Leu Cys 35 40 45 gat ttt cct ctg cgt tcg cac
ctc gaa ccc acc aac cat gcc atc att 192Asp Phe Pro Leu Arg Ser His
Leu Glu Pro Thr Asn His Ala Ile Ile 50 55 60 cag acg ttg gtc aac
tcc atg gca cca gac gcg gcg ccg gcc tcc tgc 240Gln Thr Leu Val Asn
Ser Met Ala Pro Asp Ala Ala Pro Ala Ser Cys 65 70 75 80 tgt gtc ccg
gcg cgc ctc agc ccc atc agc atc ttg tac tat gat gcc 288Cys Val Pro
Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Tyr Asp Ala 85 90 95 gcc
aac aac gtt gtc tac aag caa tac gag gac atg gtg gtg gag gcc 336Ala
Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu Ala 100 105
110 tgt ggg tgt cgc 348Cys Gly Cys Arg 115 39116PRTArtificial
SequenceSynthetic Construct 39Met Gln Ala Lys His Lys Gln Arg Lys
Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 Arg His Pro Leu Tyr Val Asp
Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Leu
Asp Tyr Glu Ala Tyr His Cys Glu Gly Leu Cys 35 40 45 Asp Phe Pro
Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Ile Ile 50 55 60 Gln
Thr Leu Val Asn Ser Met Ala Pro Asp Ala Ala Pro Ala Ser Cys 65 70
75 80 Cys Val Pro Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Tyr Asp
Ala 85 90 95 Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val
Val Glu Ala 100 105 110 Cys Gly Cys Arg 115 40330DNAArtificial
SequenceChimeric polypeptide 40atg caa gcc aaa cac aaa cag cgg aaa
cgc ctt aag tcc agc tgt aag 48Met Gln Ala Lys His Lys Gln Arg Lys
Arg Leu Lys Ser Ser Cys Lys 1 5 10 15 aga cac cct ttg tac gtg gac
ttc agt gac gtg ggg tgg aat gac tgg 96Arg His Pro Leu Tyr Val Asp
Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 att att gca ccc aaa
aga tat aag gcc aat tac tgc tct gga gag tgt 144Ile Ile Ala Pro Lys
Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys 35 40 45 gaa ttt gta
ttt tta caa aaa tac cct cac act cat ctt gtg cac caa 192Glu Phe Val
Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln 50 55 60 gca
aac ccc aga ggt tca gca ggc ccc tgc tgt act ccc aca aag atg 240Ala
Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met 65 70
75 80 tct cca atc aat atg cta tat ttt aat ggc aaa gaa caa ata ata
tat 288Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln Ile Ile
Tyr 85 90 95 ggg aaa att cca gcc atg gta gta gat cgc tgt ggg tgc
tca 330Gly Lys Ile Pro Ala Met Val Val Asp Arg Cys Gly Cys Ser 100
105 110 41110PRTArtificial SequenceSynthetic Construct 41Met Gln
Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys 1 5 10 15
Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20
25 30 Ile Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu
Cys 35 40 45 Glu Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu
Val His Gln 50 55 60 Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys
Thr Pro Thr Lys Met 65 70 75 80 Ser Pro Ile Asn Met Leu Tyr Phe Asn
Gly Lys Glu Gln Ile Ile Tyr 85 90 95 Gly Lys Ile Pro Ala Met Val
Val Asp Arg Cys Gly Cys Ser 100 105 110 42330DNAhomo
sapiensCDS(1)..(330) 42cag tgg att gaa cct cgg aat tgc gcc agg aga
tac ctc aag gta gac 48Gln Trp Ile Glu Pro Arg Asn Cys Ala Arg Arg
Tyr Leu Lys Val Asp 1 5 10 15 ttt gca gat att ggc tgg agt gaa tgg
att atc tcc ccc aag tcc ttt 96Phe Ala Asp Ile Gly Trp Ser Glu Trp
Ile Ile Ser Pro Lys Ser Phe 20 25 30 gat gcc tat tat tgc tct gga
gca tgc cag ttc ccc atg cca aag tct 144Asp Ala Tyr Tyr Cys Ser Gly
Ala Cys Gln Phe Pro Met Pro Lys Ser 35 40 45 ttg aag cca tca aat
cat gct acc atc cag agt ata gtg aga gct gtg 192Leu Lys Pro Ser Asn
His Ala Thr Ile Gln Ser Ile Val Arg Ala Val 50 55 60 ggg gtc gtt
cct ggg att cct gag cct tgc tgt gta cca gaa aag atg 240Gly Val Val
Pro Gly Ile Pro Glu Pro Cys Cys Val Pro Glu Lys Met 65 70 75 80 tcc
tca ctc agt att tta ttc ttt gat gaa aat aag aat gta gtg ctt 288Ser
Ser Leu Ser Ile Leu Phe Phe Asp Glu Asn Lys Asn Val Val Leu 85 90
95 aaa gta tac cct aac atg aca gta gag tct tgc gct tgc aga 330Lys
Val Tyr Pro Asn Met Thr Val Glu Ser Cys Ala Cys Arg 100 105 110
43110PRThomo sapiens 43Gln Trp Ile Glu Pro Arg Asn Cys Ala Arg Arg
Tyr Leu Lys Val Asp 1 5 10 15 Phe Ala Asp Ile Gly Trp Ser Glu Trp
Ile Ile Ser Pro Lys Ser Phe 20 25 30 Asp Ala Tyr Tyr Cys Ser Gly
Ala Cys Gln Phe Pro Met Pro Lys Ser 35 40 45 Leu Lys Pro Ser Asn
His Ala Thr Ile Gln Ser Ile Val Arg Ala Val 50 55 60 Gly Val Val
Pro Gly Ile Pro Glu Pro Cys Cys Val Pro Glu Lys Met 65 70 75 80 Ser
Ser Leu Ser Ile Leu Phe Phe Asp Glu Asn Lys Asn Val Val Leu 85 90
95 Lys Val Tyr Pro Asn Met Thr Val Glu Ser Cys Ala Cys Arg 100 105
110 44348DNAHomo sapiensCDS(1)..(348) 44agc cct aag cat cac tca cag
cgg gcc agg aag aag aat aag aac tgc 48Ser Pro Lys His His Ser Gln
Arg Ala Arg Lys Lys Asn Lys Asn Cys 1 5 10 15 cgg cgc cac tcg ctc
tat gtg gac ttc agc gat gtg ggc tgg aat gac 96Arg Arg His Ser Leu
Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp 20 25 30 tgg att gtg
gcc cca cca ggc tac cag gcc ttc tac tgc cat ggg gac 144Trp Ile Val
Ala Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly Asp 35 40 45 tgc
ccc ttt cca ctg gct gac cac ctc aac tca acc aac cat gcc att 192Cys
Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile 50 55
60 gtg cag acc ctg gtc aat tct gtc aat tcc agt atc ccc aaa gcc tgt
240Val Gln Thr Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys Ala Cys
65 70 75 80 tgt gtg ccc act gaa ctg agt gcc atc tcc atg ctg tac ctg
gat gag 288Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu
Asp Glu 85 90 95 tat gat aag gtg gta ctg aaa aat tat cag gag atg
gta gta gag gga 336Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met
Val Val Glu Gly 100 105 110 tgt ggg tgc cgc 348Cys Gly Cys Arg 115
45116PRTHomo sapiens 45Ser Pro Lys His His Ser Gln Arg Ala Arg Lys
Lys Asn Lys Asn Cys 1 5 10 15 Arg Arg His Ser Leu Tyr Val Asp Phe
Ser Asp Val Gly Trp Asn Asp 20 25 30 Trp Ile Val Ala Pro Pro Gly
Tyr Gln Ala Phe Tyr Cys His Gly Asp 35 40 45 Cys Pro Phe Pro Leu
Ala Asp His Leu Asn Ser Thr Asn His Ala Ile 50 55 60 Val Gln Thr
Leu Val Asn Ser Val Asn Ser Ser Ile Pro Lys Ala Cys 65 70 75 80 Cys
Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu 85 90
95 Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu Gly
100 105 110 Cys Gly Cys Arg 115 46414DNAHomo sapiensCDS(1)..(414)
46gca gcc aac aaa cga aaa aat caa aac cgc aat aaa tcc agc tct cat
48Ala Ala Asn Lys Arg Lys Asn Gln Asn Arg Asn Lys Ser Ser Ser His 1
5 10 15 cag gac tcc tcc aga atg tcc agt gtt gga gat tat aac aca agt
gag 96Gln Asp Ser Ser Arg Met Ser Ser Val Gly Asp Tyr Asn Thr Ser
Glu 20 25 30 caa aaa caa gcc tgt aag aag cac gaa ctc tat gtg agc
ttc cgg gat 144Gln Lys Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser
Phe Arg Asp 35 40 45 ctg gga tgg cag gac tgg att ata gca cca gaa
gga tac gct gca ttt 192Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu
Gly Tyr Ala Ala Phe 50 55 60 tat tgt gat gga gaa tgt tct ttt cca
ctt aac gcc cat atg aat gcc 240Tyr Cys Asp Gly Glu Cys Ser Phe Pro
Leu Asn Ala His Met Asn Ala 65 70 75 80 acc aac cac gct ata gtt cag
act ctg gtt cat ctg atg ttt cct gac 288Thr Asn His Ala Ile Val Gln
Thr Leu Val His Leu Met Phe Pro Asp 85 90 95 cac gta cca aag cct
tgt tgt gct cca acc aaa tta aat gcc atc tct 336His Val Pro Lys Pro
Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser 100 105 110 gtt ctg tac
ttt gat gac agc tcc aat gtc att ttg aaa aaa tat aga 384Val Leu Tyr
Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg 115 120 125 aat
atg gta gta cgc tca tgt ggc tgc cac 414Asn Met Val Val Arg Ser Cys
Gly Cys His 130 135 47138PRTHomo sapiens 47Ala Ala Asn Lys Arg Lys
Asn Gln Asn Arg Asn Lys Ser Ser Ser His 1 5 10 15 Gln Asp Ser Ser
Arg Met Ser Ser Val Gly Asp Tyr Asn Thr Ser Glu 20 25 30 Gln Lys
Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp 35 40 45
Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Phe 50
55 60 Tyr Cys Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn
Ala 65 70 75 80 Thr Asn His Ala Ile Val Gln Thr Leu Val His Leu Met
Phe Pro Asp 85 90 95 His Val Pro Lys Pro Cys Cys Ala Pro Thr Lys
Leu Asn Ala Ile Ser 100 105 110 Val Leu Tyr Phe Asp Asp Ser Ser Asn
Val Ile Leu Lys Lys Tyr Arg 115 120 125 Asn Met Val Val Arg Ser Cys
Gly Cys His 130 135 48396DNAHomo sapiensCDS(1)..(396) 48caa cag agt
cgt aat cgc tct acc cag tcc cag gac gtg gcg cgg gtc 48Gln Gln Ser
Arg Asn Arg Ser Thr Gln Ser Gln Asp Val Ala Arg Val 1 5 10 15 tcc
agt gct tca gat tac aac agc agt gaa ttg aaa aca gcc tgc agg 96Ser
Ser Ala Ser Asp Tyr Asn Ser Ser Glu Leu Lys Thr Ala Cys Arg 20 25
30 aag cat gag ctg tat gtg agt ttc caa gac ctg gga tgg cag gac tgg
144Lys His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Gln Asp Trp
35 40 45 atc att gca ccc aag ggc tat gct gcc aat tac tgt gat gga
gaa tgc 192Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly
Glu Cys 50 55 60 tcc ttc cca ctc aac gca cac atg aat gca acc aac
cac gcg att gtg 240Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn
His Ala Ile Val 65 70 75 80 cag acc ttg gtt cac ctt atg aac ccc gag
tat gtc ccc aaa ccg tgc 288Gln Thr Leu Val His Leu Met Asn Pro Glu
Tyr Val Pro Lys Pro Cys 85 90 95 tgt gcg cca act aag cta aat gcc
atc tcg gtt ctt tac ttt gat gac 336Cys Ala Pro Thr Lys Leu Asn Ala
Ile Ser Val Leu Tyr Phe Asp Asp 100 105 110 aac tcc aat gtc att ctg
aaa aaa tac agg aat atg gtt gta aga gct 384Asn Ser Asn Val Ile Leu
Lys Lys Tyr Arg Asn Met Val Val Arg Ala 115 120 125 tgt gga tgc cac
396Cys Gly Cys His 130 49132PRTHomo sapiens 49Gln Gln Ser Arg Asn
Arg Ser Thr Gln Ser Gln Asp Val Ala Arg Val 1 5 10 15 Ser Ser Ala
Ser Asp Tyr Asn Ser Ser Glu Leu Lys Thr Ala Cys Arg 20 25 30 Lys
His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Gln Asp Trp 35 40
45 Ile Ile Ala Pro
Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly Glu Cys 50 55 60 Ser Phe
Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala Ile Val 65 70 75 80
Gln Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys Pro Cys 85
90 95 Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp
Asp 100 105 110 Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val
Val Arg Ala 115 120 125 Cys Gly Cys His 130 50417DNAHomo
sapiensCDS(1)..(417) 50tcc acg ggg agc aaa cag cgc agc cag aac cgc
tcc aag acg ccc aag 48Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 aac cag gaa gcc ctg cgg atg gcc aac
gtg gca gag aac agc agc agc 96Asn Gln Glu Ala Leu Arg Met Ala Asn
Val Ala Glu Asn Ser Ser Ser 20 25 30 gac cag agg cag gcc tgt aag
aag cac gag ctg tat gtc agc ttc cga 144Asp Gln Arg Gln Ala Cys Lys
Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 45 gac ctg ggc tgg cag
gac tgg atc atc gcg cct gaa ggc tac gcc gcc 192Asp Leu Gly Trp Gln
Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 tac tac tgt
gag ggg gag tgt gcc ttc cct ctg aac tcc tac atg aac 240Tyr Tyr Cys
Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 gcc
acc aac cac gcc atc gtg cag acg ctg gtc cac ttc atc aac ccg 288Ala
Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90
95 gaa acg gtg ccc aag ccc tgc tgt gcg ccc acg cag ctc aat gcc atc
336Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile
100 105 110 tcc gtc ctc tac ttc gat gac agc tcc aac gtc atc ctg aag
aaa tac 384Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys
Lys Tyr 115 120 125 aga aac atg gtg gtc cgg gcc tgt ggc tgc cac
417Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 51139PRTHomo
sapiens 51Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr
Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu
Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu
Leu Tyr Val Ser Phe Arg 35 40 45 Asp Leu Gly Trp Gln Asp Trp Ile
Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 60 Tyr Tyr Cys Glu Gly Glu
Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 80 Ala Thr Asn His
Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Glu Thr
Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile 100 105 110
Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115
120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135
52417DNAHomo sapiensCDS(1)..(417) 52gca gtg agg cca ctg agg agg agg
cag ccg aag aaa agc aac gag ctg 48Ala Val Arg Pro Leu Arg Arg Arg
Gln Pro Lys Lys Ser Asn Glu Leu 1 5 10 15 ccg cag gcc aac cga ctc
cca ggg atc ttt gat gac gtc cac ggc tcc 96Pro Gln Ala Asn Arg Leu
Pro Gly Ile Phe Asp Asp Val His Gly Ser 20 25 30 cac ggc cgg cag
gtc tgc cgt cgg cac gag ctc tac gtc agc ttc cag 144His Gly Arg Gln
Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gln 35 40 45 gac ctc
ggc tgg ctg gac tgg gtc atc gct ccc caa ggc tac tcg gcc 192Asp Leu
Gly Trp Leu Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala 50 55 60
tat tac tgt gag ggg gag tgc tcc ttc cca ctg gac tcc tgc atg aat
240Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn
65 70 75 80 gcc acc aac cac gcc atc ctg cag tcc ctg gtg cac ctg atg
atg cca 288Ala Thr Asn His Ala Ile Leu Gln Ser Leu Val His Leu Met
Met Pro 85 90 95 gac gca gtc ccc aag gcg tgc tgt gca ccc acc aag
ctg agc gcc acc 336Asp Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys
Leu Ser Ala Thr 100 105 110 tct gtg ctc tac tat gac agc agc aac aat
gtc atc ctg cgc aag cac 384Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn
Val Ile Leu Arg Lys His 115 120 125 cgc aac atg gtg gtc aag gcc tgc
ggc tgc cac 417Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135
53139PRTHomo sapiens 53Ala Val Arg Pro Leu Arg Arg Arg Gln Pro Lys
Lys Ser Asn Glu Leu 1 5 10 15 Pro Gln Ala Asn Arg Leu Pro Gly Ile
Phe Asp Asp Val His Gly Ser 20 25 30 His Gly Arg Gln Val Cys Arg
Arg His Glu Leu Tyr Val Ser Phe Gln 35 40 45 Asp Leu Gly Trp Leu
Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala 50 55 60 Tyr Tyr Cys
Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn 65 70 75 80 Ala
Thr Asn His Ala Ile Leu Gln Ser Leu Val His Leu Met Met Pro 85 90
95 Asp Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr
100 105 110 Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg
Lys His 115 120 125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130
135 54333DNAHomo sapiensCDS(1)..(333) 54agc gcc ggg gct ggc agc cac
tgt caa aag acc tcc ctg cgg gta aac 48Ser Ala Gly Ala Gly Ser His
Cys Gln Lys Thr Ser Leu Arg Val Asn 1 5 10 15 ttc gag gac atc ggc
tgg gac agc tgg atc att gca ccc aag gag tat 96Phe Glu Asp Ile Gly
Trp Asp Ser Trp Ile Ile Ala Pro Lys Glu Tyr 20 25 30 gaa gcc tac
gag tgt aag ggc ggc tgc ttc ttc ccc ttg gct gac gat 144Glu Ala Tyr
Glu Cys Lys Gly Gly Cys Phe Phe Pro Leu Ala Asp Asp 35 40 45 gtg
acg ccg acg aaa cac gct atc gtg cag acc ctg gtg cat ctc aag 192Val
Thr Pro Thr Lys His Ala Ile Val Gln Thr Leu Val His Leu Lys 50 55
60 ttc ccc aca aag gtg ggc aag gcc tgc tgt gtg ccc acc aaa ctg agc
240Phe Pro Thr Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu Ser
65 70 75 80 ccc atc tcc gtc ctc tac aag gat gac atg ggg gtg ccc acc
ctc aag 288Pro Ile Ser Val Leu Tyr Lys Asp Asp Met Gly Val Pro Thr
Leu Lys 85 90 95 tac cat tac gag ggc atg agc gtg gca gag tgt ggg
tgc agg tag 333Tyr His Tyr Glu Gly Met Ser Val Ala Glu Cys Gly Cys
Arg 100 105 110 55110PRTHomo sapiens 55Ser Ala Gly Ala Gly Ser His
Cys Gln Lys Thr Ser Leu Arg Val Asn 1 5 10 15 Phe Glu Asp Ile Gly
Trp Asp Ser Trp Ile Ile Ala Pro Lys Glu Tyr 20 25 30 Glu Ala Tyr
Glu Cys Lys Gly Gly Cys Phe Phe Pro Leu Ala Asp Asp 35 40 45 Val
Thr Pro Thr Lys His Ala Ile Val Gln Thr Leu Val His Leu Lys 50 55
60 Phe Pro Thr Lys Val Gly Lys Ala Cys Cys Val Pro Thr Lys Leu Ser
65 70 75 80 Pro Ile Ser Val Leu Tyr Lys Asp Asp Met Gly Val Pro Thr
Leu Lys 85 90 95 Tyr His Tyr Glu Gly Met Ser Val Ala Glu Cys Gly
Cys Arg 100 105 110 56324DNAHomo sapiensCDS(1)..(324) 56aac gcc aaa
gga aac tac tgt aag agg acc ccg ctc tac atc gac ttc 48Asn Ala Lys
Gly Asn Tyr Cys Lys Arg Thr Pro Leu Tyr Ile Asp Phe 1 5 10 15 aag
gag att ggg tgg gac tcc tgg atc atc gct ccg cct gga tac gaa 96Lys
Glu Ile Gly Trp Asp Ser Trp Ile Ile Ala Pro Pro Gly Tyr Glu 20 25
30 gcc tat gaa tgc cgt ggt gtt tgt aac tac ccc ctg gca gag cat ctc
144Ala Tyr Glu Cys Arg Gly Val Cys Asn Tyr Pro Leu Ala Glu His Leu
35 40 45 aca ccc aca aag cat gca att atc cag gcc ttg gtc cac ctc
aag aat 192Thr Pro Thr Lys His Ala Ile Ile Gln Ala Leu Val His Leu
Lys Asn 50 55 60 tcc cag aaa gct tcc aaa gcc tgc tgt gtg ccc aca
aag cta gag ccc 240Ser Gln Lys Ala Ser Lys Ala Cys Cys Val Pro Thr
Lys Leu Glu Pro 65 70 75 80 atc tcc atc ctc tat tta gac aaa ggc gtc
gtc acc tac aag ttt aaa 288Ile Ser Ile Leu Tyr Leu Asp Lys Gly Val
Val Thr Tyr Lys Phe Lys 85 90 95 tac gaa ggc atg gcc gtc tcc gaa
tgt ggc tgt aga 324Tyr Glu Gly Met Ala Val Ser Glu Cys Gly Cys Arg
100 105 57108PRTHomo sapiens 57Asn Ala Lys Gly Asn Tyr Cys Lys Arg
Thr Pro Leu Tyr Ile Asp Phe 1 5 10 15 Lys Glu Ile Gly Trp Asp Ser
Trp Ile Ile Ala Pro Pro Gly Tyr Glu 20 25 30 Ala Tyr Glu Cys Arg
Gly Val Cys Asn Tyr Pro Leu Ala Glu His Leu 35 40 45 Thr Pro Thr
Lys His Ala Ile Ile Gln Ala Leu Val His Leu Lys Asn 50 55 60 Ser
Gln Lys Ala Ser Lys Ala Cys Cys Val Pro Thr Lys Leu Glu Pro 65 70
75 80 Ile Ser Ile Leu Tyr Leu Asp Lys Gly Val Val Thr Tyr Lys Phe
Lys 85 90 95 Tyr Glu Gly Met Ala Val Ser Glu Cys Gly Cys Arg 100
105 58375DNAHomo sapiensCDS(1)..(375) 58caa gca gat ggt atc tca gct
gag gtt act gcc tct tcc tca aaa cat 48Gln Ala Asp Gly Ile Ser Ala
Glu Val Thr Ala Ser Ser Ser Lys His 1 5 10 15 agc ggg cct gaa aat
aac cag tgt tcc ctc cac cct ttc caa atc agc 96Ser Gly Pro Glu Asn
Asn Gln Cys Ser Leu His Pro Phe Gln Ile Ser 20 25 30 ttc cgc cag
ctg ggt tgg gat cac tgg atc att gct ccc cct ttc tac 144Phe Arg Gln
Leu Gly Trp Asp His Trp Ile Ile Ala Pro Pro Phe Tyr 35 40 45 acc
cca aac tac tgt aaa gga act tgt ctc cga gta cta cgc gat ggt 192Thr
Pro Asn Tyr Cys Lys Gly Thr Cys Leu Arg Val Leu Arg Asp Gly 50 55
60 ctc aat tcc ccc aat cac gcc att att cag aac ctt atc aat cag ttg
240Leu Asn Ser Pro Asn His Ala Ile Ile Gln Asn Leu Ile Asn Gln Leu
65 70 75 80 gtg gac cag agt gtc ccc cgg ccc tcc tgt gtc ccg tat aag
tat gtt 288Val Asp Gln Ser Val Pro Arg Pro Ser Cys Val Pro Tyr Lys
Tyr Val 85 90 95 cca att agt gtc ctt atg att gag gca aat ggg agt
att ttg tac aag 336Pro Ile Ser Val Leu Met Ile Glu Ala Asn Gly Ser
Ile Leu Tyr Lys 100 105 110 gag tat gag ggt atg att gct gag tct tgt
aca tgc aga 375Glu Tyr Glu Gly Met Ile Ala Glu Ser Cys Thr Cys Arg
115 120 125 59125PRTHomo sapiens 59Gln Ala Asp Gly Ile Ser Ala Glu
Val Thr Ala Ser Ser Ser Lys His 1 5 10 15 Ser Gly Pro Glu Asn Asn
Gln Cys Ser Leu His Pro Phe Gln Ile Ser 20 25 30 Phe Arg Gln Leu
Gly Trp Asp His Trp Ile Ile Ala Pro Pro Phe Tyr 35 40 45 Thr Pro
Asn Tyr Cys Lys Gly Thr Cys Leu Arg Val Leu Arg Asp Gly 50 55 60
Leu Asn Ser Pro Asn His Ala Ile Ile Gln Asn Leu Ile Asn Gln Leu 65
70 75 80 Val Asp Gln Ser Val Pro Arg Pro Ser Cys Val Pro Tyr Lys
Tyr Val 85 90 95 Pro Ile Ser Val Leu Met Ile Glu Ala Asn Gly Ser
Ile Leu Tyr Lys 100 105 110 Glu Tyr Glu Gly Met Ile Ala Glu Ser Cys
Thr Cys Arg 115 120 125 60357DNAHomo sapiensCDS(1)..(357) 60gac gcc
gaa ccc gtg ttg ggc ggc ggc ccc ggg ggc gct tgt cgc gcg 48Asp Ala
Glu Pro Val Leu Gly Gly Gly Pro Gly Gly Ala Cys Arg Ala 1 5 10 15
cgg cgg ctg tac gtg agc ttc cgc gag gtg ggc tgg cac cgc tgg gtc
96Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His Arg Trp Val
20 25 30 atc gcg ccg cgc ggc ttc ctg gcc aac tac tgc cag ggt cag
tgc gcg 144Ile Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gln Gly Gln
Cys Ala 35 40 45 ctg ccc gtc gcg ctg tcg ggg tcc ggg ggg ccg ccg
gcg ctc aac cac 192Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro
Ala Leu Asn His 50 55 60 gct gtg ctg cgc gcg ctc atg cac gcg gcc
gcc ccg gga gcc gcc gac 240Ala Val Leu Arg Ala Leu Met His Ala Ala
Ala Pro Gly Ala Ala Asp 65 70 75 80 ctg ccc tgc tgc gtg ccc gcg cgc
ctg tcg ccc atc tcc gtg ctc ttc 288Leu Pro Cys Cys Val Pro Ala Arg
Leu Ser Pro Ile Ser Val Leu Phe 85 90 95 ttt gac aac agc gac aac
gtg gtg ctg cgg cag tat gag gac atg gtg 336Phe Asp Asn Ser Asp Asn
Val Val Leu Arg Gln Tyr Glu Asp Met Val 100 105 110 gtg gac gag tgc
ggc tgc cgc 357Val Asp Glu Cys Gly Cys Arg 115 61119PRTHomo sapiens
61Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly Ala Cys Arg Ala 1
5 10 15 Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His Arg Trp
Val 20 25 30 Ile Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gln Gly
Gln Cys Ala 35 40 45 Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro
Pro Ala Leu Asn His 50 55 60 Ala Val Leu Arg Ala Leu Met His Ala
Ala Ala Pro Gly Ala Ala Asp 65 70 75 80 Leu Pro Cys Cys Val Pro Ala
Arg Leu Ser Pro Ile Ser Val Leu Phe 85 90 95 Phe Asp Asn Ser Asp
Asn Val Val Leu Arg Gln Tyr Glu Asp Met Val 100 105 110 Val Asp Glu
Cys Gly Cys Arg 115 62342DNAHomo sapiensCDS(1)..(342) 62gca gcc atc
cct gtc ccc aag ctt tct tgt aag aac ctc tgc cac cgt 48Ala Ala Ile
Pro Val Pro Lys Leu Ser Cys Lys Asn Leu Cys His Arg 1 5 10 15 cac
cag cta ttc att aac ttc cgg gac ctg ggt tgg cac aag tgg atc 96His
Gln Leu Phe Ile Asn Phe Arg Asp Leu Gly Trp His Lys Trp Ile 20 25
30 att gcc ccc aag ggg ttc atg gca aat tac tgc cat gga gag tgt ccc
144Ile Ala Pro Lys Gly Phe Met Ala Asn Tyr Cys His Gly Glu Cys Pro
35 40 45 ttc tca ctg acc atc tct ctc aac agc tcc aat tat gct ttc
atg caa 192Phe Ser Leu Thr Ile Ser Leu Asn Ser Ser Asn Tyr Ala Phe
Met Gln 50 55 60 gcc ctg atg cat gcc gtt gac cca gag atc ccc cag
gct gtg tgt atc 240Ala Leu Met His Ala Val Asp Pro
Glu Ile Pro Gln Ala Val Cys Ile 65 70 75 80 ccc acc aag ctg tct ccc
att tcc atg ctc tac cag gac aat aat gac 288Pro Thr Lys Leu Ser Pro
Ile Ser Met Leu Tyr Gln Asp Asn Asn Asp 85 90 95 aat gtc att cta
cga cat tat gaa gac atg gta gtc gat gaa tgt ggg 336Asn Val Ile Leu
Arg His Tyr Glu Asp Met Val Val Asp Glu Cys Gly 100 105 110 tgt ggg
342Cys Gly 63114PRTHomo sapiens 63Ala Ala Ile Pro Val Pro Lys Leu
Ser Cys Lys Asn Leu Cys His Arg 1 5 10 15 His Gln Leu Phe Ile Asn
Phe Arg Asp Leu Gly Trp His Lys Trp Ile 20 25 30 Ile Ala Pro Lys
Gly Phe Met Ala Asn Tyr Cys His Gly Glu Cys Pro 35 40 45 Phe Ser
Leu Thr Ile Ser Leu Asn Ser Ser Asn Tyr Ala Phe Met Gln 50 55 60
Ala Leu Met His Ala Val Asp Pro Glu Ile Pro Gln Ala Val Cys Ile 65
70 75 80 Pro Thr Lys Leu Ser Pro Ile Ser Met Leu Tyr Gln Asp Asn
Asn Asp 85 90 95 Asn Val Ile Leu Arg His Tyr Glu Asp Met Val Val
Asp Glu Cys Gly 100 105 110 Cys Gly 64360DNAHomo
sapiensCDS(1)..(360) 64gcc cca ctg gcc act cgc cag ggc aag cga ccc
agc aag aac ctt aag 48Ala Pro Leu Ala Thr Arg Gln Gly Lys Arg Pro
Ser Lys Asn Leu Lys 1 5 10 15 gct cgc tgc agt cgg aag gca ctg cat
gtc aac ttc aag gac atg ggc 96Ala Arg Cys Ser Arg Lys Ala Leu His
Val Asn Phe Lys Asp Met Gly 20 25 30 tgg gac gac tgg atc atc gca
ccc ctt gag tac gag gct ttc cac tgc 144Trp Asp Asp Trp Ile Ile Ala
Pro Leu Glu Tyr Glu Ala Phe His Cys 35 40 45 gag ggg ctg tgc gag
ttc cca ttg cgc tcc cac ctg gag ccc acg aat 192Glu Gly Leu Cys Glu
Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50 55 60 cat gca gtc
atc cag acc ctg atg aac tcc atg gac ccc gag tcc aca 240His Ala Val
Ile Gln Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr 65 70 75 80 cca
ccc acc tgc tgt gtg ccc acg cgg ctg agt ccc atc agc atc ctc 288Pro
Pro Thr Cys Cys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile Leu 85 90
95 ttc att gac tct gcc aac aac gtg gtg tat aag cag tat gag gac atg
336Phe Ile Asp Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met
100 105 110 gtc gtg gag tcg tgt ggc tgc agg 360Val Val Glu Ser Cys
Gly Cys Arg 115 120 65120PRTHomo sapiens 65Ala Pro Leu Ala Thr Arg
Gln Gly Lys Arg Pro Ser Lys Asn Leu Lys 1 5 10 15 Ala Arg Cys Ser
Arg Lys Ala Leu His Val Asn Phe Lys Asp Met Gly 20 25 30 Trp Asp
Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Phe His Cys 35 40 45
Glu Gly Leu Cys Glu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50
55 60 His Ala Val Ile Gln Thr Leu Met Asn Ser Met Asp Pro Glu Ser
Thr 65 70 75 80 Pro Pro Thr Cys Cys Val Pro Thr Arg Leu Ser Pro Ile
Ser Ile Leu 85 90 95 Phe Ile Asp Ser Ala Asn Asn Val Val Tyr Lys
Gln Tyr Glu Asp Met 100 105 110 Val Val Glu Ser Cys Gly Cys Arg 115
120 66360DNAHomo sapiensCDS(1)..(360) 66acg gcc ttc gcc agt cgc cat
ggc aag cgg cac ggc aag aag tcc agg 48Thr Ala Phe Ala Ser Arg His
Gly Lys Arg His Gly Lys Lys Ser Arg 1 5 10 15 cta cgc tgc agc aag
aag ccc ctg cac gtg aac ttc aag gag ctg ggc 96Leu Arg Cys Ser Lys
Lys Pro Leu His Val Asn Phe Lys Glu Leu Gly 20 25 30 tgg gac gac
tgg att atc gcg ccc ctg gag tac gag gcc tat cac tgc 144Trp Asp Asp
Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Tyr His Cys 35 40 45 gag
ggt gta tgc gac ttc ccg ctg cgc tcg cac ctg gag ccc acc aac 192Glu
Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn 50 55
60 cac gcc atc atc cag acg ctg atg aac tcc atg gac ccc ggc tcc acc
240His Ala Ile Ile Gln Thr Leu Met Asn Ser Met Asp Pro Gly Ser Thr
65 70 75 80 ccg ccc agc tgc tgc gtg ccc acc aaa ttg act ccc atc agc
att cta 288Pro Pro Ser Cys Cys Val Pro Thr Lys Leu Thr Pro Ile Ser
Ile Leu 85 90 95 tac atc gac gcg ggc aat aat gtg gtc tac aag cag
tac gag gac atg 336Tyr Ile Asp Ala Gly Asn Asn Val Val Tyr Lys Gln
Tyr Glu Asp Met 100 105 110 gtg gtg gag tcg tgc ggc tgc agg 360Val
Val Glu Ser Cys Gly Cys Arg 115 120 67120PRTHomo sapiens 67Thr Ala
Phe Ala Ser Arg His Gly Lys Arg His Gly Lys Lys Ser Arg 1 5 10 15
Leu Arg Cys Ser Lys Lys Pro Leu His Val Asn Phe Lys Glu Leu Gly 20
25 30 Trp Asp Asp Trp Ile Ile Ala Pro Leu Glu Tyr Glu Ala Tyr His
Cys 35 40 45 Glu Gly Val Cys Asp Phe Pro Leu Arg Ser His Leu Glu
Pro Thr Asn 50 55 60 His Ala Ile Ile Gln Thr Leu Met Asn Ser Met
Asp Pro Gly Ser Thr 65 70 75 80 Pro Pro Ser Cys Cys Val Pro Thr Lys
Leu Thr Pro Ile Ser Ile Leu 85 90 95 Tyr Ile Asp Ala Gly Asn Asn
Val Val Tyr Lys Gln Tyr Glu Asp Met 100 105 110 Val Val Glu Ser Cys
Gly Cys Arg 115 120 68387DNAHomo sapiensCDS(1)..(387) 68acg gcg ttg
gcc ggg acg cgg aca tcg cag ggc agc ggc ggg ggc gcg 48Thr Ala Leu
Ala Gly Thr Arg Thr Ser Gln Gly Ser Gly Gly Gly Ala 1 5 10 15 ggc
cgg ggc cac ggg cgc agg ggc cgg agc cgc tgc agc cgc aag ccg 96Gly
Arg Gly His Gly Arg Arg Gly Arg Ser Arg Cys Ser Arg Lys Pro 20 25
30 ttg cac gtg gac ttc aag gag ctc ggc tgg gac gac tgg atc atc gcg
144Leu His Val Asp Phe Lys Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala
35 40 45 ccg ctg gac tac gag gcg tac cac tgc gag ggc ctt tgc gac
ttc cct 192Pro Leu Asp Tyr Glu Ala Tyr His Cys Glu Gly Leu Cys Asp
Phe Pro 50 55 60 ttg cgt tcg cac ctc gag ccc acc aac cat gcc atc
att cag acg ctg 240Leu Arg Ser His Leu Glu Pro Thr Asn His Ala Ile
Ile Gln Thr Leu 65 70 75 80 ctc aac tcc atg gca cca gac gcg gcg ccg
gcc tcc tgc tgt gtg cca 288Leu Asn Ser Met Ala Pro Asp Ala Ala Pro
Ala Ser Cys Cys Val Pro 85 90 95 gcg cgc ctc agc ccc atc agc atc
ctc tac atc gac gcc gcc aac aac 336Ala Arg Leu Ser Pro Ile Ser Ile
Leu Tyr Ile Asp Ala Ala Asn Asn 100 105 110 gtt gtc tac aag caa tac
gag gac atg gtg gtg gag gcc tgc ggc tgc 384Val Val Tyr Lys Gln Tyr
Glu Asp Met Val Val Glu Ala Cys Gly Cys 115 120 125 agg 387Arg
69129PRTHomo sapiens 69Thr Ala Leu Ala Gly Thr Arg Thr Ser Gln Gly
Ser Gly Gly Gly Ala 1 5 10 15 Gly Arg Gly His Gly Arg Arg Gly Arg
Ser Arg Cys Ser Arg Lys Pro 20 25 30 Leu His Val Asp Phe Lys Glu
Leu Gly Trp Asp Asp Trp Ile Ile Ala 35 40 45 Pro Leu Asp Tyr Glu
Ala Tyr His Cys Glu Gly Leu Cys Asp Phe Pro 50 55 60 Leu Arg Ser
His Leu Glu Pro Thr Asn His Ala Ile Ile Gln Thr Leu 65 70 75 80 Leu
Asn Ser Met Ala Pro Asp Ala Ala Pro Ala Ser Cys Cys Val Pro 85 90
95 Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Ile Asp Ala Ala Asn Asn
100 105 110 Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val Glu Ala Cys
Gly Cys 115 120 125 Arg 70327DNAHomo sapiensCDS(1)..(327) 70gat ttt
ggt ctt gac tgt gat gag cac tca aca gaa tca cga tgc tgt 48Asp Phe
Gly Leu Asp Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys 1 5 10 15
cgt tac cct cta act gtg gat ttt gaa gct ttt gga tgg gat tgg att
96Arg Tyr Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile
20 25 30 atc gct cct aaa aga tat aag gcc aat tac tgc tct gga gag
tgt gaa 144Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu
Cys Glu 35 40 45 ttt gta ttt tta caa aaa tat cct cat act cat ctg
gta cac caa gca 192Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu
Val His Gln Ala 50 55 60 aac ccc aga ggt tca gca ggc cct tgc tgt
act ccc aca aag atg tct 240Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys
Thr Pro Thr Lys Met Ser 65 70 75 80 cca att aat atg cta tat ttt aat
ggc aaa gaa caa ata ata tat ggg 288Pro Ile Asn Met Leu Tyr Phe Asn
Gly Lys Glu Gln Ile Ile Tyr Gly 85 90 95 aaa att cca gcg atg gta
gta gac cgc tgt ggg tgc tca 327Lys Ile Pro Ala Met Val Val Asp Arg
Cys Gly Cys Ser 100 105 71109PRTHomo sapiens 71Asp Phe Gly Leu Asp
Cys Asp Glu His Ser Thr Glu Ser Arg Cys Cys 1 5 10 15 Arg Tyr Pro
Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile
Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu Cys Glu 35 40
45 Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His Gln Ala
50 55 60 Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys
Met Ser 65 70 75 80 Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu Gln
Ile Ile Tyr Gly 85 90 95 Lys Ile Pro Ala Met Val Val Asp Arg Cys
Gly Cys Ser 100 105 72405DNAHomo sapiensCDS(1)..(405) 72ggt cag gaa
act gtc agt tct gaa ttg aag aag ccc ttg ggc cca gct 48Gly Gln Glu
Thr Val Ser Ser Glu Leu Lys Lys Pro Leu Gly Pro Ala 1 5 10 15 tcc
ttc aat ctg agt gaa tac ttc aga caa ttt ctt ctt ccc caa aat 96Ser
Phe Asn Leu Ser Glu Tyr Phe Arg Gln Phe Leu Leu Pro Gln Asn 20 25
30 gag tgt gag ctc cat gac ttt aga ctt agc ttt agt cag ctg aag tgg
144Glu Cys Glu Leu His Asp Phe Arg Leu Ser Phe Ser Gln Leu Lys Trp
35 40 45 gac aac tgg att gtg gct ccg cac agg tac aac cct cga tac
tgt aaa 192Asp Asn Trp Ile Val Ala Pro His Arg Tyr Asn Pro Arg Tyr
Cys Lys 50 55 60 ggg gac tgt cca agg gca gtt gga cat cgg tat ggc
tct cca gtt cac 240Gly Asp Cys Pro Arg Ala Val Gly His Arg Tyr Gly
Ser Pro Val His 65 70 75 80 acc atg gta cag aac atc atc tat gag aag
ctg gac tcc tca gtg cca 288Thr Met Val Gln Asn Ile Ile Tyr Glu Lys
Leu Asp Ser Ser Val Pro 85 90 95 aga ccg tca tgt gta cct gcc aaa
tac agc ccc ttg agt gtt ttg acc 336Arg Pro Ser Cys Val Pro Ala Lys
Tyr Ser Pro Leu Ser Val Leu Thr 100 105 110 att gag ccc gat ggc tca
att gcc tat aaa gag tac gaa gat atg ata 384Ile Glu Pro Asp Gly Ser
Ile Ala Tyr Lys Glu Tyr Glu Asp Met Ile 115 120 125 gct aca aag tgc
acc tgt cgt 405Ala Thr Lys Cys Thr Cys Arg 130 135 73135PRTHomo
sapiens 73Gly Gln Glu Thr Val Ser Ser Glu Leu Lys Lys Pro Leu Gly
Pro Ala 1 5 10 15 Ser Phe Asn Leu Ser Glu Tyr Phe Arg Gln Phe Leu
Leu Pro Gln Asn 20 25 30 Glu Cys Glu Leu His Asp Phe Arg Leu Ser
Phe Ser Gln Leu Lys Trp 35 40 45 Asp Asn Trp Ile Val Ala Pro His
Arg Tyr Asn Pro Arg Tyr Cys Lys 50 55 60 Gly Asp Cys Pro Arg Ala
Val Gly His Arg Tyr Gly Ser Pro Val His 65 70 75 80 Thr Met Val Gln
Asn Ile Ile Tyr Glu Lys Leu Asp Ser Ser Val Pro 85 90 95 Arg Pro
Ser Cys Val Pro Ala Lys Tyr Ser Pro Leu Ser Val Leu Thr 100 105 110
Ile Glu Pro Asp Gly Ser Ile Ala Tyr Lys Glu Tyr Glu Asp Met Ile 115
120 125 Ala Thr Lys Cys Thr Cys Arg 130 135 74360DNAHomo
sapiensCDS(1)..(360) 74aag acg atg cag aaa gcc cgg agg aag cag tgg
gat gag ccg agg gtg 48Lys Thr Met Gln Lys Ala Arg Arg Lys Gln Trp
Asp Glu Pro Arg Val 1 5 10 15 tgc tcc cgg agg tac ctg aag gtg gac
ttc gca gac atc ggc tgg aat 96Cys Ser Arg Arg Tyr Leu Lys Val Asp
Phe Ala Asp Ile Gly Trp Asn 20 25 30 gaa tgg ata atc tca ccg aaa
tct ttt gat gcc tac tac tgc gcg gga 144Glu Trp Ile Ile Ser Pro Lys
Ser Phe Asp Ala Tyr Tyr Cys Ala Gly 35 40 45 gca tgt gag ttc ccc
atg cct aag atc gtt cgt cca tcc aac cat gcc 192Ala Cys Glu Phe Pro
Met Pro Lys Ile Val Arg Pro Ser Asn His Ala 50 55 60 acc atc cag
agc att gtc agg gct gtg ggc atc atc cct ggc atc cca 240Thr Ile Gln
Ser Ile Val Arg Ala Val Gly Ile Ile Pro Gly Ile Pro 65 70 75 80 gag
ccc tgc tgt gtt ccc gat aag atg aac tcc ctt ggg gtc ctc ttc 288Glu
Pro Cys Cys Val Pro Asp Lys Met Asn Ser Leu Gly Val Leu Phe 85 90
95 ctg gat gag aat cgg aat gtg gtt ctg aag gtg tac ccc aac atg tcc
336Leu Asp Glu Asn Arg Asn Val Val Leu Lys Val Tyr Pro Asn Met Ser
100 105 110 gtg gac acc tgt gcc tgc cgg tga 360Val Asp Thr Cys Ala
Cys Arg 115 75119PRTHomo sapiens 75Lys Thr Met Gln Lys Ala Arg Arg
Lys Gln Trp Asp Glu Pro Arg Val 1 5 10 15 Cys Ser Arg Arg Tyr Leu
Lys Val Asp Phe Ala Asp Ile Gly Trp Asn 20 25 30 Glu Trp Ile Ile
Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala Gly 35 40 45 Ala Cys
Glu Phe Pro Met Pro Lys Ile Val Arg Pro Ser Asn His Ala 50 55 60
Thr Ile Gln Ser Ile Val Arg Ala Val Gly Ile Ile Pro Gly Ile Pro 65
70 75 80 Glu Pro Cys Cys Val Pro Asp Lys Met Asn Ser Leu Gly Val
Leu Phe 85 90 95 Leu Asp Glu Asn Arg Asn Val Val Leu Lys Val Tyr
Pro Asn Met Ser 100 105 110 Val Asp Thr Cys Ala Cys Arg 115
76327DNAHomo sapiensCDS(1)..(327) 76aac ctg ggt ctg gac tgc gac gag
cac tca agc gag tcc cgc tgc tgc 48Asn Leu Gly Leu Asp Cys Asp Glu
His Ser Ser Glu Ser Arg Cys Cys 1 5 10 15 cga tat ccc ctc aca gtg
gac ttt gag gct ttc ggc tgg gac tgg atc 96Arg Tyr Pro Leu Thr Val
Asp Phe Glu Ala Phe Gly Trp Asp Trp Ile 20 25 30 atc gca cct aag
cgc tac aag gcc aac tac tgc tcc ggc cag tgc gag 144Ile Ala Pro Lys
Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln Cys Glu 35 40 45 tac atg
ttc atg caa aaa tat ccg cat acc cat ttg gtg cag cag gcc 192Tyr Met
Phe Met Gln Lys Tyr Pro His Thr His Leu Val Gln Gln Ala 50 55 60
aat cca aga ggc tct gct ggg ccc tgt tgt acc ccc acc aag atg tcc
240Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser
65 70 75 80 cca atc aac atg ctc tac ttc aat gac aag cag cag att atc
tac ggc 288Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile
Tyr Gly 85 90 95 aag atc cct ggc atg gtg gtg gat cgc tgt ggc tgc
tct 327Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly Cys Ser 100 105
77109PRTHomo sapiens 77Asn Leu Gly Leu Asp Cys Asp Glu His Ser Ser
Glu Ser Arg Cys Cys 1 5 10 15 Arg Tyr Pro Leu Thr Val Asp Phe Glu
Ala Phe Gly Trp Asp Trp Ile 20 25 30 Ile Ala Pro Lys Arg Tyr Lys
Ala Asn Tyr Cys Ser Gly Gln Cys Glu 35 40 45 Tyr Met Phe Met Gln
Lys Tyr Pro His Thr His Leu Val Gln Gln Ala 50 55 60 Asn Pro Arg
Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr Lys Met Ser 65 70 75 80 Pro
Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln Gln Ile Ile Tyr Gly 85 90
95 Lys Ile Pro Gly Met Val Val Asp Arg Cys Gly Cys Ser 100 105
78336DNAHomo sapiensCDS(1)..(336) 78gcg cgc aac ggg gac cac tgt ccg
ctc ggg ccc ggg cgt tgc tgc cgt 48Ala Arg Asn Gly Asp His Cys Pro
Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 ctg cac acg gtc cgc gcg
tcg ctg gaa gac ctg ggc tgg gcc gat tgg 96Leu His Thr Val Arg Ala
Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30 gtg ctg tcg cca
cgg gag gtg caa gtg acc atg tgc atc ggc gcg tgc 144Val Leu Ser Pro
Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35 40 45 ccg agc
cag ttc cgg gcg gca aac atg cac gcg cag atc aag acg agc 192Pro Ser
Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr Ser 50 55 60
ctg cac cgc ctg aag ccc gac acg gtg cca gcg ccc tgc tgc gtg ccc
240Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys Cys Val Pro
65 70 75 80 gcc agc tac aat ccc atg gtg ctc att caa aag acc gac acc
ggg gtg 288Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys Thr Asp Thr
Gly Val 85 90 95 tcg ctc cag acc tat gat gac ttg tta gcc aaa gac
tgc cac tgc ata 336Ser Leu Gln Thr Tyr Asp Asp Leu Leu Ala Lys Asp
Cys His Cys Ile 100 105 110 79112PRTHomo sapiens 79Ala Arg Asn Gly
Asp His Cys Pro Leu Gly Pro Gly Arg Cys Cys Arg 1 5 10 15 Leu His
Thr Val Arg Ala Ser Leu Glu Asp Leu Gly Trp Ala Asp Trp 20 25 30
Val Leu Ser Pro Arg Glu Val Gln Val Thr Met Cys Ile Gly Ala Cys 35
40 45 Pro Ser Gln Phe Arg Ala Ala Asn Met His Ala Gln Ile Lys Thr
Ser 50 55 60 Leu His Arg Leu Lys Pro Asp Thr Val Pro Ala Pro Cys
Cys Val Pro 65 70 75 80 Ala Ser Tyr Asn Pro Met Val Leu Ile Gln Lys
Thr Asp Thr Gly Val 85 90 95 Ser Leu Gln Thr Tyr Asp Asp Leu Leu
Ala Lys Asp Cys His Cys Ile 100 105 110 80330DNAHomo
sapiensCDS(1)..(330) 80cat cac ttg cca gac aga agt caa ctg tgt cgg
aag gtc aag ttc cag 48His His Leu Pro Asp Arg Ser Gln Leu Cys Arg
Lys Val Lys Phe Gln 1 5 10 15 gtg gac ttc aac ctg atc gga tgg ggc
tcc tgg atc atc tac ccc aag 96Val Asp Phe Asn Leu Ile Gly Trp Gly
Ser Trp Ile Ile Tyr Pro Lys 20 25 30 cag tac aac gcc tat cgc tgt
gag ggc gag tgt cct aat cct gtt ggg 144Gln Tyr Asn Ala Tyr Arg Cys
Glu Gly Glu Cys Pro Asn Pro Val Gly 35 40 45 gag gag ttt cat ccg
acc aac cat gca tac atc cag agt ctg ctg aaa 192Glu Glu Phe His Pro
Thr Asn His Ala Tyr Ile Gln Ser Leu Leu Lys 50 55 60 cgt tac cag
ccc cac cga gtc cct tcc act tgt tgt gcc cca gtg aag 240Arg Tyr Gln
Pro His Arg Val Pro Ser Thr Cys Cys Ala Pro Val Lys 65 70 75 80 acc
aag ccg ctg agc atg ctg tat gtg gat aat ggc aga gtg ctc cta 288Thr
Lys Pro Leu Ser Met Leu Tyr Val Asp Asn Gly Arg Val Leu Leu 85 90
95 gat cac cat aaa gac atg atc gtg gaa gaa tgt ggg tgc ctc 330Asp
His His Lys Asp Met Ile Val Glu Glu Cys Gly Cys Leu 100 105 110
81110PRTHomo sapiens 81His His Leu Pro Asp Arg Ser Gln Leu Cys Arg
Lys Val Lys Phe Gln 1 5 10 15 Val Asp Phe Asn Leu Ile Gly Trp Gly
Ser Trp Ile Ile Tyr Pro Lys 20 25 30 Gln Tyr Asn Ala Tyr Arg Cys
Glu Gly Glu Cys Pro Asn Pro Val Gly 35 40 45 Glu Glu Phe His Pro
Thr Asn His Ala Tyr Ile Gln Ser Leu Leu Lys 50 55 60 Arg Tyr Gln
Pro His Arg Val Pro Ser Thr Cys Cys Ala Pro Val Lys 65 70 75 80 Thr
Lys Pro Leu Ser Met Leu Tyr Val Asp Asn Gly Arg Val Leu Leu 85 90
95 Asp His His Lys Asp Met Ile Val Glu Glu Cys Gly Cys Leu 100 105
110 82345DNAHomo sapiensCDS(1)..(345) 82ggc ctg gag tgc gac ggc aag
gtc aac atc tgc tgt aag aaa cag ttc 48Gly Leu Glu Cys Asp Gly Lys
Val Asn Ile Cys Cys Lys Lys Gln Phe 1 5 10 15 ttt gtc agt ttc aag
gac atc ggc tgg aat gac tgg atc att gct ccc 96Phe Val Ser Phe Lys
Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro 20 25 30 tct ggc tat
cat gcc aac tac tgc gag ggt gag tgc ccg agc cat ata 144Ser Gly Tyr
His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile 35 40 45 gca
ggc acg tcc ggg tcc tca ctg tcc ttc cac tca aca gtc atc aac 192Ala
Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn 50 55
60 cac tac gca tgc ggc cat agc ccc ttt gcc aac ctc aaa tcg tgc tgt
240His Tyr Ala Cys Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys Cys
65 70 75 80 gtg ccc acc aag ctg aga ccc atg tcc atg ttg tac tat gat
gat ggt 288Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp
Asp Gly 85 90 95 caa aac atc atc aaa aag gac att cag aac atg atc
gtg gag gag tgc 336Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile
Val Glu Glu Cys 100 105 110 ggg tgc tcc 345Gly Cys Ser 115
83115PRTHomo sapiens 83Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys
Cys Lys Lys Gln Phe 1 5 10 15 Phe Val Ser Phe Lys Asp Ile Gly Trp
Asn Asp Trp Ile Ile Ala Pro 20 25 30 Ser Gly Tyr His Ala Asn Tyr
Cys Glu Gly Glu Cys Pro Ser His Ile 35 40 45 Ala Gly Thr Ser Gly
Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn 50 55 60 His Tyr Ala
Cys Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys Cys 65 70 75 80 Val
Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp Gly 85 90
95 Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu Cys
100 105 110 Gly Cys Ser 115 84345DNAHomo sapiensCDS(1)..(345) 84ggc
ctg gag tgc gat ggc cgg acc aac ctc tgt tgc agg caa cag ttc 48Gly
Leu Glu Cys Asp Gly Arg Thr Asn Leu Cys Cys Arg Gln Gln Phe 1 5 10
15 ttc att gac ttc cgc ctc atc ggc tgg aac gac tgg atc ata gca ccc
96Phe Ile Asp Phe Arg Leu Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro
20 25 30 acc ggc tac tac ggc aac tac tgt gag ggc agc tgc cca gcc
tac ctg 144Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser Cys Pro Ala
Tyr Leu 35 40 45 gca ggg gtc ccc ggc tct gcc tcc tcc ttc cac acg
gct gtg gtg aac 192Ala Gly Val Pro Gly Ser Ala Ser Ser Phe His Thr
Ala Val Val Asn 50 55 60 cag tac cgc atg cgg ggt ctg aac ccc ggc
acg gtg aac tcc tgc tgc 240Gln Tyr Arg Met Arg Gly Leu Asn Pro Gly
Thr Val Asn Ser Cys Cys 65 70 75 80 att ccc acc aag ctg agc acc atg
tcc atg ctg tac ttc gat gat gag 288Ile Pro Thr Lys Leu Ser Thr Met
Ser Met Leu Tyr Phe Asp Asp Glu 85 90 95 tac aac atc gtc aag cgg
gac gtg ccc aac atg att gtg gag gag tgc 336Tyr Asn Ile Val Lys Arg
Asp Val Pro Asn Met Ile Val Glu Glu Cys 100 105 110 ggc tgc gcc
345Gly Cys Ala 115 85115PRTHomo sapiens 85Gly Leu Glu Cys Asp Gly
Arg Thr Asn Leu Cys Cys Arg Gln Gln Phe 1 5 10 15 Phe Ile Asp Phe
Arg Leu Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro 20 25 30 Thr Gly
Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser Cys Pro Ala Tyr Leu 35 40 45
Ala Gly Val Pro Gly Ser Ala Ser Ser Phe His Thr Ala Val Val Asn 50
55 60 Gln Tyr Arg Met Arg Gly Leu Asn Pro Gly Thr Val Asn Ser Cys
Cys 65 70 75 80 Ile Pro Thr Lys Leu Ser Thr Met Ser Met Leu Tyr Phe
Asp Asp Glu 85 90 95 Tyr Asn Ile Val Lys Arg Asp Val Pro Asn Met
Ile Val Glu Glu Cys 100 105 110 Gly Cys Ala 115 86348DNAHomo
sapiensCDS(1)..(348) 86ggc atc gac tgc caa gga ggg tcc agg atg tgc
tgt cga caa gag ttt 48Gly Ile Asp Cys Gln Gly Gly Ser Arg Met Cys
Cys Arg Gln Glu Phe 1 5 10 15 ttt gtg gac ttc cgt gag att ggc tgg
cac gac tgg atc atc cag cct 96Phe Val Asp Phe Arg Glu Ile Gly Trp
His Asp Trp Ile Ile Gln Pro 20 25 30 gag ggc tac gcc atg aac ttc
tgc ata ggg cag tgc cca cta cac ata 144Glu Gly Tyr Ala Met Asn Phe
Cys Ile Gly Gln Cys Pro Leu His Ile 35 40 45 gca ggc atg cct ggt
att gct gcc tcc ttt cac act gca gtg ctc aat 192Ala Gly Met Pro Gly
Ile Ala Ala Ser Phe His Thr Ala Val Leu Asn 50 55 60 ctt ctc aag
gcc aac aca gct gca ggc acc act gga ggg ggc tca tgc 240Leu Leu Lys
Ala Asn Thr Ala Ala Gly Thr Thr Gly Gly Gly Ser Cys 65 70 75 80 tgt
gta ccc acg gcc cgg cgc ccc ctg tct ctg ctc tat tat gac agg 288Cys
Val Pro Thr Ala Arg Arg Pro Leu Ser Leu Leu Tyr Tyr Asp Arg 85 90
95 gac agc aac att gtc aag act gac ata cct gac atg gta gta gag gcc
336Asp Ser Asn Ile Val Lys Thr Asp Ile Pro Asp Met Val Val Glu Ala
100 105 110 tgt ggg tgc agt 348Cys Gly Cys Ser 115 87116PRTHomo
sapiens 87Gly Ile Asp Cys Gln Gly Gly Ser Arg Met Cys Cys Arg Gln
Glu Phe 1 5 10 15 Phe Val Asp Phe Arg Glu Ile Gly Trp His Asp Trp
Ile Ile Gln Pro 20 25 30 Glu Gly Tyr Ala Met Asn Phe Cys Ile Gly
Gln Cys Pro Leu His Ile 35 40 45 Ala Gly Met Pro Gly Ile Ala Ala
Ser Phe His Thr Ala Val Leu Asn 50 55 60 Leu Leu Lys Ala Asn Thr
Ala Ala Gly Thr Thr Gly Gly Gly Ser Cys 65 70 75 80 Cys Val Pro Thr
Ala Arg Arg Pro Leu Ser Leu Leu Tyr Tyr Asp Arg 85 90 95 Asp Ser
Asn Ile Val Lys Thr Asp Ile Pro Asp Met Val Val Glu Ala 100 105 110
Cys Gly Cys Ser 115 88342DNAHomo sapiensCDS(1)..(342) 88acc ccc acc
tgt gag cct gcg acc ccc tta tgt tgc agg cga gac cat 48Thr Pro Thr
Cys Glu Pro Ala Thr Pro Leu Cys Cys Arg Arg Asp His 1 5 10 15 tac
gta gac ttc cag gaa ctg gga tgg cgg gac tgg ata ctg cag ccc 96Tyr
Val Asp Phe Gln Glu Leu Gly Trp Arg Asp Trp Ile Leu Gln Pro 20 25
30 gag ggg tac cag ctg aat tac tgc agt ggg cag tgc cct ccc cac ctg
144Glu Gly Tyr Gln Leu Asn Tyr Cys Ser Gly Gln Cys Pro Pro His Leu
35 40 45 gct ggc agc cca ggc att gct gcc tct ttc cat tct gcc gtc
ttc agc 192Ala Gly Ser Pro Gly Ile Ala Ala Ser Phe His Ser Ala Val
Phe Ser 50 55 60 ctc ctc aaa gcc aac aat cct tgg cct gcc agt acc
tcc tgt tgt gtc 240Leu Leu Lys Ala Asn Asn Pro Trp Pro Ala Ser Thr
Ser Cys Cys Val 65 70 75 80 cct act gcc cga agg ccc ctc tct ctc ctc
tac ctg gat cat aat ggc 288Pro Thr Ala Arg Arg Pro Leu Ser Leu Leu
Tyr Leu Asp His Asn Gly 85 90 95 aat gtg gtc aag acg gat gtg cca
gat atg gtg gtg gag gcc tgt ggc 336Asn Val Val Lys Thr Asp Val Pro
Asp Met Val Val Glu Ala Cys Gly 100 105 110 tgc agc 342Cys Ser
89114PRTHomo sapiens 89Thr Pro Thr Cys Glu Pro Ala Thr Pro Leu Cys
Cys Arg Arg Asp His 1 5 10 15 Tyr Val Asp Phe Gln Glu Leu Gly Trp
Arg Asp Trp Ile Leu Gln Pro 20 25 30 Glu Gly Tyr Gln Leu Asn Tyr
Cys Ser Gly Gln Cys Pro Pro His Leu 35 40 45 Ala Gly Ser Pro Gly
Ile Ala Ala Ser Phe His Ser Ala Val Phe Ser 50 55 60 Leu Leu Lys
Ala Asn Asn Pro Trp Pro Ala Ser Thr Ser Cys Cys Val 65 70 75 80 Pro
Thr Ala Arg Arg Pro Leu Ser Leu Leu Tyr Leu Asp His Asn Gly 85 90
95 Asn Val Val Lys Thr Asp Val Pro Asp Met Val Val Glu Ala Cys Gly
100 105 110 Cys Ser 90402DNAHomo sapiensCDS(1)..(402) 90tca act ccc
ctg atg tcc tgg cct tgg tct ccc tct gct ctg cgc ctg 48Ser Thr Pro
Leu Met Ser Trp Pro Trp Ser Pro Ser Ala Leu Arg Leu 1 5
10 15 ctg cag agg cct ccg gag gaa ccg gct gcc cat gcc aac tgc cac
aga 96Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala His Ala Asn Cys His
Arg 20 25 30 gta gca ctg aac atc tcc ttc cag gag ctg ggc tgg gaa
cgg tgg atc 144Val Ala Leu Asn Ile Ser Phe Gln Glu Leu Gly Trp Glu
Arg Trp Ile 35 40 45 gtg tac cct ccc agt ttc atc ttc cac tac tgt
cat ggt ggt tgt ggg 192Val Tyr Pro Pro Ser Phe Ile Phe His Tyr Cys
His Gly Gly Cys Gly 50 55 60 ctg cac atc cca cca aac ctg tcc ctt
cca gtc cct ggg gct ccc cct 240Leu His Ile Pro Pro Asn Leu Ser Leu
Pro Val Pro Gly Ala Pro Pro 65 70 75 80 acc cca gcc cag ccc tac tcc
ttg ctg cca ggg gcc cag ccc tgc tgt 288Thr Pro Ala Gln Pro Tyr Ser
Leu Leu Pro Gly Ala Gln Pro Cys Cys 85 90 95 gct gct ctc cca ggg
acc atg agg ccc cta cat gtc cgc acc acc tcg 336Ala Ala Leu Pro Gly
Thr Met Arg Pro Leu His Val Arg Thr Thr Ser 100 105 110 gat gga ggt
tac tct ttc aag tat gag aca gtg ccc aac ctt ctc acg 384Asp Gly Gly
Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn Leu Leu Thr 115 120 125 cag
cac tgt gct tgt atc 402Gln His Cys Ala Cys Ile 130 91134PRTHomo
sapiens 91Ser Thr Pro Leu Met Ser Trp Pro Trp Ser Pro Ser Ala Leu
Arg Leu 1 5 10 15 Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala His Ala
Asn Cys His Arg 20 25 30 Val Ala Leu Asn Ile Ser Phe Gln Glu Leu
Gly Trp Glu Arg Trp Ile 35 40 45 Val Tyr Pro Pro Ser Phe Ile Phe
His Tyr Cys His Gly Gly Cys Gly 50 55 60 Leu His Ile Pro Pro Asn
Leu Ser Leu Pro Val Pro Gly Ala Pro Pro 65 70 75 80 Thr Pro Ala Gln
Pro Tyr Ser Leu Leu Pro Gly Ala Gln Pro Cys Cys 85 90 95 Ala Ala
Leu Pro Gly Thr Met Arg Pro Leu His Val Arg Thr Thr Ser 100 105 110
Asp Gly Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn Leu Leu Thr 115
120 125 Gln His Cys Ala Cys Ile 130 92336DNAHomo
sapiensCDS(1)..(336) 92gcc ctg gac acc aac tat tgc ttc agc tcc acg
gag aag aac tgc tgc 48Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr
Glu Lys Asn Cys Cys 1 5 10 15 gtg cgg cag ctg tac att gac ttc cgc
aag gac ctc ggc tgg aag tgg 96Val Arg Gln Leu Tyr Ile Asp Phe Arg
Lys Asp Leu Gly Trp Lys Trp 20 25 30 atc cac gag ccc aag ggc tac
cat gcc aac ttc tgc ctc ggg ccc tgc 144Ile His Glu Pro Lys Gly Tyr
His Ala Asn Phe Cys Leu Gly Pro Cys 35 40 45 ccc tac att tgg agc
ctg gac acg cag tac agc aag gtc ctg gcc ctg 192Pro Tyr Ile Trp Ser
Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu 50 55 60 tac aac cag
cat aac ccg ggc gcc tcg gcg gcg ccg tgc tgc gtg ccg 240Tyr Asn Gln
His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro 65 70 75 80 cag
gcg ctg gag ccg ctg ccc atc gtg tac tac gtg ggc cgc aag ccc 288Gln
Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro 85 90
95 aag gtg gag cag ctg tcc aac atg atc gtg cgc tcc tgc aag tgc agc
336Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser
100 105 110 93112PRTHomo sapiens 93Ala Leu Asp Thr Asn Tyr Cys Phe
Ser Ser Thr Glu Lys Asn Cys Cys 1 5 10 15 Val Arg Gln Leu Tyr Ile
Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp 20 25 30 Ile His Glu Pro
Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys 35 40 45 Pro Tyr
Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu 50 55 60
Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro 65
70 75 80 Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg
Lys Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser
Cys Lys Cys Ser 100 105 110 94336DNAHomo sapiensCDS(1)..(336) 94gct
ttg gat gcg gcc tat tgc ttt aga aat gtg cag gat aat tgc tgc 48Ala
Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn Cys Cys 1 5 10
15 cta cgt cca ctt tac att gat ttc aag agg gat cta ggg tgg aaa tgg
96Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp Leu Gly Trp Lys Trp
20 25 30 ata cac gaa ccc aaa ggg tac aat gcc aac ttc tgt gct gga
gca tgc 144Ile His Glu Pro Lys Gly Tyr Asn Ala Asn Phe Cys Ala Gly
Ala Cys 35 40 45 ccg tat tta tgg agt tca gac act cag cac agc agg
gtc ctg agc tta 192Pro Tyr Leu Trp Ser Ser Asp Thr Gln His Ser Arg
Val Leu Ser Leu 50 55 60 tat aat acc ata aat cca gaa gca tct gct
tct cct tgc tgc gtg tcc 240Tyr Asn Thr Ile Asn Pro Glu Ala Ser Ala
Ser Pro Cys Cys Val Ser 65 70 75 80 caa gat tta gaa cct cta acc att
ctc tac tac att ggc aaa aca ccc 288Gln Asp Leu Glu Pro Leu Thr Ile
Leu Tyr Tyr Ile Gly Lys Thr Pro 85 90 95 aag att gaa cag ctt tct
aat atg att gta aag tct tgc aaa tgc agc 336Lys Ile Glu Gln Leu Ser
Asn Met Ile Val Lys Ser Cys Lys Cys Ser 100 105 110 95112PRTHomo
sapiens 95Ala Leu Asp Ala Ala Tyr Cys Phe Arg Asn Val Gln Asp Asn
Cys Cys 1 5 10 15 Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp Leu
Gly Trp Lys Trp 20 25 30 Ile His Glu Pro Lys Gly Tyr Asn Ala Asn
Phe Cys Ala Gly Ala Cys 35 40 45 Pro Tyr Leu Trp Ser Ser Asp Thr
Gln His Ser Arg Val Leu Ser Leu 50 55 60 Tyr Asn Thr Ile Asn Pro
Glu Ala Ser Ala Ser Pro Cys Cys Val Ser 65 70 75 80 Gln Asp Leu Glu
Pro Leu Thr Ile Leu Tyr Tyr Ile Gly Lys Thr Pro 85 90 95 Lys Ile
Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys Lys Cys Ser 100 105 110
96336DNAHomo sapiensCDS(1)..(336) 96gct ttg gac acc aat tac tgc ttc
cgc aac ttg gag gag aac tgc tgt 48Ala Leu Asp Thr Asn Tyr Cys Phe
Arg Asn Leu Glu Glu Asn Cys Cys 1 5 10 15 gtg cgc ccc ctc tac att
gac ttc cga cag gat ctg ggc tgg aag tgg 96Val Arg Pro Leu Tyr Ile
Asp Phe Arg Gln Asp Leu Gly Trp Lys Trp 20 25 30 gtc cat gaa cct
aag ggc tac tat gcc aac ttc tgc tca ggc cct tgc 144Val His Glu Pro
Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly Pro Cys 35 40 45 cca tac
ctc cgc agt gca gac aca acc cac agc acg gtg ctg gga ctg 192Pro Tyr
Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val Leu Gly Leu 50 55 60
tac aac act ctg aac cct gaa gca tct gcc tcg cct tgc tgc gtg ccc
240Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys Cys Val Pro
65 70 75 80 cag gac ctg gag ccc ctg acc atc ctg tac tat gtt ggg agg
acc ccc 288Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Val Gly Arg
Thr Pro 85 90 95 aaa gtg gag cag ctc tcc aac atg gtg gtg aag tct
tgt aaa tgt agc 336Lys Val Glu Gln Leu Ser Asn Met Val Val Lys Ser
Cys Lys Cys Ser 100 105 110 97112PRTHomo sapiens 97Ala Leu Asp Thr
Asn Tyr Cys Phe Arg Asn Leu Glu Glu Asn Cys Cys 1 5 10 15 Val Arg
Pro Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly Trp Lys Trp 20 25 30
Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly Pro Cys 35
40 45 Pro Tyr Leu Arg Ser Ala Asp Thr Thr His Ser Thr Val Leu Gly
Leu 50 55 60 Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro Cys
Cys Val Pro 65 70 75 80 Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr Tyr
Val Gly Arg Thr Pro 85 90 95 Lys Val Glu Gln Leu Ser Asn Met Val
Val Lys Ser Cys Lys Cys Ser 100 105 110 9830PRTArtificial
SequenceSection 1 of BMP-2 98Gln Ala Lys His Lys Gln Arg Lys Arg
Leu Lys Ser Ser Cys Lys Arg1 5 10 15 His Pro Leu Tyr Val Asp Phe
Ser Asp Val Gly Trp Asn Asp 20 25 30 9918PRTArtificial
SequenceSection 2 of BMP-2 99Trp Ile Val Ala Pro Pro Gly Tyr His
Ala Phe Tyr Cys His Gly Glu1 5 10 15 Cys Pro10020PRTArtificial
SequenceSection 3 of BMP-2 100Phe Pro Leu Ala Asp His Leu Asn Ser
Thr Asn His Ala Ile Val Gln1 5 10 15 Thr Leu Val Asn 20
10113PRTArtificial SequenceSection 4 of BMP-2 101Ser Val Asn Ser
Lys Ile Pro Lys Ala Cys Cys Val Pro1 5 10 10212PRTArtificial
SequenceSection 5 of BMP-2 102Thr Glu Leu Ser Ala Ile Ser Met Leu
Tyr Tyr Asp1 5 10 10321PRTArtificial SequenceSection 6 of BMP-2
103Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val Val Glu1
5 10 15 Gly Cys Gly Cys Arg 20 10429PRTArtificial SequenceSection 1
of activin-betaA 104Arg Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys
Cys Lys Lys Gln1 5 10 15 Phe Phe Val Ser Phe Lys Asp Ile Gly Trp
Asn Asp Trp 20 25 10518PRTArtificial SequenceSection 2 of
activin-betaA 105Trp Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr
Cys Glu Gly Glu1 5 10 15 Cys Pro10619PRTArtificial SequenceSection
3 of activin-betaA 106Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu
Ser Phe His Ser Thr1 5 10 15 Leu Val Asn10719PRTArtificial
SequenceSection 4 of activin-betaA 107His Tyr Arg Met Arg Gly His
Ser Pro Phe Ala Asn Leu Lys Ser Cys1 5 10 15 Cys Val
Pro10812PRTArtificial SequenceSection 5 of activin-betaA 108Thr Lys
Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp1 5 10 10921PRTArtificial
SequenceSection 6 of activin-betaA 109Asp Gly Gln Asn Ile Ile Lys
Lys Asp Ile Gln Asn Met Ile Val Glu1 5 10 15 Glu Cys Gly Cys Ser 20
11032PRTArtificial SequenceBMP-2 Sequence 110Gln Ala Lys His Lys
Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys Arg1 5 10 15 His Pro Leu
Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Ile Ile 20 25 30
111345DNAArtificial SequenceAB2-016 111atgcaagcca aacacaaaca
gcggaagcgt cttaagtcca gctgcaaaag gcaccctttg 60tatgtggact tcagtgatgt
ggggtggaat gactggatca ttgctccctc tggctatcat 120gccaactact
gcgagggaga atgccctttt cctctggctg atcatctgaa ctccactaat
180cacgccattg ttcagacgtt ggtcaactct gttaactcta agattcctaa
ggcatgctgt 240gtcccgacag aactcagtgc tatctcgatg ctgtaccttg
acgagaatga aaaggttgta 300ttaaagaact atcaggacat ggttgtggag
ggttgcgggt gtcgt 345112115PRTArtificial SequenceAB2-016 112Met Gln
Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15
Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20
25 30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu
Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His
Ala Ile Val 50 55 60 Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile
Pro Lys Ala Cys Cys65 70 75 80 Val Pro Thr Glu Leu Ser Ala Ile Ser
Met Leu Tyr Leu Asp Glu Asn 85 90 95 Glu Lys Val Val Leu Lys Asn
Tyr Gln Asp Met Val Val Glu Gly Cys 100 105 110 Gly Cys Arg 115
113363DNAArtificial SequenceAB2-017 113atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggattg tggctccccc ggggtatcac 120gccttttact
gccacggaga atgccctttt cctctggctg atcatctgaa ctccactaat
180catgccattg ttcagacgtt ggtcaaccac taccgcatgc ggggccatag
cccctttgcc 240aacctcaaat cgtgctgtgt cccgaccaag ctgagaccca
tgtccatgtt gtactatgat 300gatggtcaaa acatcatcaa aaaggacatt
cagaacatga tcgtggagga gtgtgggtgc 360tca 363114121PRTArtificial
SequenceAB2-017 114Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp
Val Gly Trp Asn Asp Trp 20 25 30 Ile Val Ala Pro Pro Gly Tyr His
Ala Phe Tyr Cys His Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp
His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val
Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala65 70 75 80 Asn Leu
Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 85 90 95
Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn 100
105 110 Met Ile Val Glu Glu Cys Gly Cys Ser 115 120
115363DNAArtificial SequenceAB2-018 115atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggattg tggctccccc ggggtatcac 120gccttttact
gccacggaga atgccctttt cctctggctg atcatctgaa ctccactaat
180catgccattg ttcagacgtt ggtcaaccac taccgcatgc ggggccatag
cccctttgcc 240aacctcaaat cgtgctgtgt cccgacagaa ctcagtgcta
tctcgatact gtaccttgac 300gagaatgaaa aggttgtatt aaagaactat
caggacatgg ttgtggaggg ttgtgggtgt 360cgc 363116121PRTArtificial
SequenceAB2-018 116Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp
Val Gly Trp Asn Asp Trp 20 25 30 Ile Val Ala Pro Pro Gly Tyr His
Ala Phe Tyr Cys His Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp
His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val
Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala65 70 75 80 Asn Leu
Lys Ser Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Ile 85 90 95
Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp 100
105 110 Met Val Val Glu Gly Cys Gly Cys Arg 115 120
117363DNAArtificial SequenceAB2-019 117atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggattg tggctccccc ggggtatcac 120gccttttact
gccacggaga atgccctttt cctctggctg atcatctgaa ctccactaat
180catgccattg ttcagacgtt ggtcaaccac taccgcatgc ggggccatag
cccctttgcc 240aacctcaaat cgtgctgtgt cccgaccaag ctgagaccca
tgtccatgtt gtactatgat 300gagaatgaaa aggttgtatt aaagaactat
caggacatgg ttgtggaggg ttgcgggtgt 360cgt 363118121PRTArtificial
SequenceAB2-019 118Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys1 5 10
15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp
20 25 30 Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly
Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn
His Ala Ile Val 50 55 60 Gln Thr Leu Val Asn His Tyr Arg Met Arg
Gly His Ser Pro Phe Ala65 70 75 80 Asn Leu Lys Ser Cys Cys Val Pro
Thr Lys Leu Arg Pro Met Ser Met 85 90 95 Leu Tyr Tyr Asp Glu Asn
Glu Lys Val Val Leu Lys Asn Tyr Gln Asp 100 105 110 Met Val Val Glu
Gly Cys Gly Cys Arg 115 120 119363DNAArtificial SequenceAB2-020
119atgcaagcca aacacaaaca gcggaaacgc cttaagtcca gctgtaagag
acaccctttg 60tacgtggact tcagtgacgt ggggtggaat gactggattg tggctccccc
ggggtatcac 120gccttttact gccacggaga atgccctttt cctctggctg
atcatctgaa ctccactaat 180catgccattg ttcagacgtt ggtcaaccac
taccgcatgc ggggccatag cccctttgcc 240aacctcaaat cgtgctgtgt
cccgacagaa ctcagtgcta tctcgatgtt gtactatgat 300gatggtcaaa
acatcatcaa aaaggacatt cagaacatga tcgtggagga gtgtgggtgc 360tca
363120121PRTArtificial SequenceAB2-020 120Met Gln Ala Lys His Lys
Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu
Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile Val
Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35 40 45
Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala Ile Val 50
55 60 Gln Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser Pro Phe
Ala65 70 75 80 Asn Leu Lys Ser Cys Cys Val Pro Thr Glu Leu Ser Ala
Ile Ser Met 85 90 95 Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys
Lys Asp Ile Gln Asn 100 105 110 Met Ile Val Glu Glu Cys Gly Cys Ser
115 120 121360DNAArtificial SequenceAB2-021 121atgcaagcca
aacacaaaca gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tatgtggact
tcagtgacgt ggggtggaat gactggattg tggctccccc ggggtatcac
120gccttttact gccacggaga atgcccttct catatagcag gcacgtccgg
gtcctcactg 180tccttccact caacgttggt caaccactac cgcatgcggg
gccatagccc ctttgccaac 240ctcaaatcgt gctgtgtccc gaccaagctg
agacccatgt ccatgttgta ccttgacgag 300aatgaaaagg ttgtattaaa
gaactatcag gacatggttg tggagggttg tgggtgtcgc 360122120PRTArtificial
SequenceAB2-021 122Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp
Val Gly Trp Asn Asp Trp 20 25 30 Ile Val Ala Pro Pro Gly Tyr His
Ala Phe Tyr Cys His Gly Glu Cys 35 40 45 Pro Ser His Ile Ala Gly
Thr Ser Gly Ser Ser Leu Ser Phe His Ser 50 55 60 Thr Leu Val Asn
His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn65 70 75 80 Leu Lys
Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu 85 90 95
Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met 100
105 110 Val Val Glu Gly Cys Gly Cys Arg 115 120 123351DNAArtificial
SequenceAB2-022 123atgcaagcca aacacaaaca gcggaaacgc cttaagtcca
gctgtaagag acaccctttg 60tacgtggact tcagtgacgt ggggtggaat gactggattg
tggctccccc ggggtatcac 120gccttttact gccacggaga atgcccttct
catatagcag gcacgtccgg gtcctcactg 180tccttccact caacgttggt
caaccactac cgcatgcggg gccatagccc ctttgccaac 240ctcaaatcgt
gctgtgtccc gacagaactc agtgctatct cgatgttgta ctatgatgag
300aatgaaaagg ttgtattaaa gaactatcag gacatggttg tggagggttg c
351124120PRTArtificial SequenceAB2-022 124Met Gln Ala Lys His Lys
Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu
Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25 30 Ile Val
Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys 35 40 45
Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser 50
55 60 Thr Leu Val Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala
Asn65 70 75 80 Leu Lys Ser Cys Cys Val Pro Thr Glu Leu Ser Ala Ile
Ser Met Leu 85 90 95 Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys
Asn Tyr Gln Asp Met 100 105 110 Val Val Glu Gly Cys Gly Cys Arg 115
120 125342DNAArtificial SequenceAB2-023 125atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggattg tggctccccc ggggtatcac 120gccttttact
gccacggaga atgcccttct catatagcag gcacgtccgg gtcctcactg
180tccttccact caacgttggt caactctgtt aactctaaga ttcctaaggc
atgctgtgtc 240ccgacagaac tcagtgctat ctcgatgctg taccttgacg
agaatgaaaa ggttgtatta 300aagaactatc aggacatggt tgtggagggt
tgcgggtgtc gt 342126114PRTArtificial SequenceAB2-023 126Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys
35 40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys
Ala Cys Cys Val65 70 75 80 Pro Thr Glu Leu Ser Ala Ile Ser Met Leu
Tyr Leu Asp Glu Asn Glu 85 90 95 Lys Val Val Leu Lys Asn Tyr Gln
Asp Met Val Val Glu Gly Cys Gly 100 105 110 Cys
Arg127342DNAArtificial SequenceAB2-024 127atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggattg tggctccccc ggggtatcac 120gccttttact
gccacggaga atgcccttct catatagcag gcacgtccgg gtcctcactg
180tccttccact caacgttggt caactctgtt aactctaaga ttcctaaggc
atgctgtgtc 240ccgacagaac tcagtgctat ctcgatgttg tactatgatg
atggtcaaaa catcatcaaa 300aaggacattc agaacatgat cgtggaggag
tgtgggtgct ca 342128114PRTArtificial SequenceAB2-024 128Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys
35 40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys
Ala Cys Cys Val65 70 75 80 Pro Thr Glu Leu Ser Ala Ile Ser Met Leu
Tyr Tyr Asp Asp Gly Gln 85 90 95 Asn Ile Ile Lys Lys Asp Ile Gln
Asn Met Ile Val Glu Glu Cys Gly 100 105 110 Cys
Ser129342DNAArtificial SequenceAB2-025 129atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggattg tggctccccc ggggtatcac 120gccttttact
gccacggaga atgcccttct catatagcag gcacgtccgg gtcctcactg
180tccttccact caacgttggt caactctgtt aactctaaga ttcctaaggc
atgctgtgtc 240ccgaccaagc tgagacccat gtccatgttg tactatgatg
atggtcaaaa catcatcaaa 300aaggacattc agaacatgat cgtggaggag
tgtgggtgct ca 342130114PRTArtificial SequenceAB2-025 130Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys
35 40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys
Ala Cys Cys Val65 70 75 80 Pro Thr Lys Leu Arg Pro Met Ser Met Leu
Tyr Tyr Asp Asp Gly Gln 85 90 95 Asn Ile Ile Lys Lys Asp Ile Gln
Asn Met Ile Val Glu Glu Cys Gly 100 105 110 Cys
Ser131342DNAArtificial SequenceAB2-026 131atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggattg tggctccccc ggggtatcac 120gccttttact
gccacggaga atgcccttct catatagcag gcacgtccgg gtcctcactg
180tccttccact caacgttggt caactctgtt aactctaaga ttcctaaggc
atgctgtgtc 240ccgaccaagc tgagacccat gtccatgttg tactatgatg
agaatgaaaa ggttgtatta 300aagaactatc aggacatggt tgtggagggt
tgcgggtgtc gt 342132114PRTArtificial SequenceAB2-026 132Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu Cys
35 40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys
Ala Cys Cys Val65 70 75 80 Pro Thr Lys Leu Arg Pro Met Ser Met Leu
Tyr Tyr Asp Glu Asn Glu 85 90 95 Lys Val Val Leu Lys Asn Tyr Gln
Asp Met Val Val Glu Gly Cys Gly 100 105 110 Cys
Arg133342DNAArtificial SequenceAB2-027 133atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggatca ttgctccctc tggctatcat 120gccaactact
gcgagggaga atgcccttct catatagcag gcacgtccgg gtcctcactg
180tccttccact caacgttggt caactctgtt aactctaaga ttcctaaggc
atgctgtgtc 240ccgacagaac tcagtgctat ctcgatgttg taccttgacg
agaatgaaaa ggttgtatta 300aagaactatc aggacatggt tgtggagggt
tgtgggtgtc gc 342134114PRTArtificial SequenceAB2-027 134Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys
35 40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys
Ala Cys Cys Val65 70 75 80 Pro Thr Glu Leu Ser Ala Ile Ser Met Leu
Tyr Leu Asp Glu Asn Glu 85 90 95 Lys Val Val Leu Lys Asn Tyr Gln
Asp Met Val Val Glu Gly Cys Gly 100 105 110 Cys
Arg135342DNAArtificial SequenceAB2-028 135atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggatca ttgctccctc tggctatcat 120gccaactact
gcgagggaga atgcccttct catatagcag gcacgtccgg gtcctcactg
180tccttccact caacgttggt caactctgtt aactctaaga ttcctaaggc
atgctgtgtc 240ccgacagaac tcaatgctat ctcgatgttg tactatgatg
atggtcaaaa catcattaaa 300aaggacattc agaacatgat cgtggaggag
tgtgggtgct ca 342136114PRTArtificial SequenceAB2-028 136Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys
35 40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys
Ala Cys Cys Val65 70 75 80 Pro Thr Glu Leu Asn Ala Ile Ser Met Leu
Tyr Tyr Asp Asp Gly Gln 85 90 95 Asn Ile Ile Lys Lys Asp Ile Gln
Asn Met Ile Val Glu Glu Cys Gly 100 105 110 Cys
Ser137342DNAArtificial SequenceAB2-030 137atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggatca ttgctccctc tggctatcat 120gccaactact
gcgagggaga atgcccttct catatagcag gcacgtccgg gtcctcactg
180tccttccact caacgttggt caactctgtt aactctaaga ttcctaaggc
atgctgtgtc 240ccgaccaagc tgagacccat gtccatgttg tactatgatg
atggtcaaaa catcatcaaa 300aaggacattc agaacatgat cgtggaggag
tgtgggtgct ca 342138114PRTArtificial SequenceAB2-030 138Met Gln Ala
Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys Lys1 5 10 15 Arg
His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp Trp 20 25
30 Ile Ile Ala Pro Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys
35 40 45 Pro Ser His Ile Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe
His Ser 50 55 60 Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys
Ala Cys Cys Val65 70 75 80 Pro Thr Lys Leu Arg Pro Met Ser Met Leu
Tyr Tyr Asp Asp Gly Gln 85 90 95 Asn Ile Ile Lys Lys Asp Ile Gln
Asn Met Ile Val Glu Glu Cys Gly 100 105 110 Cys
Ser139363DNAArtificial SequenceAB2-031 139atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggatca ttgctccctc tggctatcat 120gccaactact
gcgagggaga atgccctttt cctctggctg atcatctgaa ctccactaat
180catgccattg ttcagacgtt ggtcaaccac taccgcatgc ggggccatag
cccctttgcc 240aacctcaaat cgtgctgtgt cccgaccaag ctgagaccca
tgtccatgtt gtactatgat 300gatggtcaaa acatcatcaa aaaggacatt
cagaacatga tcgtggagga gtgtgggtgc 360tca 363140121PRTArtificial
SequenceAB2-031 140Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp
Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Ser Gly Tyr His
Ala Asn Tyr Cys Glu Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp
His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val
Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala65 70 75 80 Asn Leu
Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 85 90 95
Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn 100
105 110 Met Ile Val Glu Glu Cys Gly Cys Ser 115 120
141363DNAArtificial SequenceAB2-032 141atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggatca ttgctccctc tggctatcat 120gccaactact
gcgagggaga atgccctttt cctctggctg atcatctgaa ctccactaat
180catgccattg ttcagacgtt ggtcaaccac taccgcatgc ggggccatag
cccctttgcc 240aacctcaaat cgtgctgtgt cccgacagaa ctcagtgcta
tctcgatgct gtaccttgac 300gatggtcaaa acatcatcaa aaaggacatt
cagaacatga tcgtggagga gtgtgggtgc 360tca 363142121PRTArtificial
SequenceAB2-032 142Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp
Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Ser Gly Tyr His
Ala Asn Tyr Cys Glu Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp
His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val
Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala65 70 75 80 Asn Leu
Lys Ser Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met 85 90 95
Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn
100 105 110 Met Ile Val Glu Glu Cys Gly Cys Ser 115 120
143363DNAArtificial SequenceAB2-033 143atgcaagcca aacacaaaca
gcggaaacgc cttaagtcca gctgtaagag acaccctttg 60tacgtggact tcagtgacgt
ggggtggaat gactggatca ttgctccctc tggctatcat 120gccaactact
gcgagggaga atgccctttt cctctggctg atcatctgaa ctctactaat
180catgccattg ttcagacgtt ggtcaaccac taccgcatgc ggggccatag
cccctttgcc 240aacctcaaat cgtgctgtgt cccgacagaa ctcagtgcta
tctcgatgct gtaccttgac 300gagaatgaaa aggttgtatt aaagaactat
caggacatgg ttgtggaggg ttgcgggtgt 360cgt 363144121PRTArtificial
SequenceAB2-033 144Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp
Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Ser Gly Tyr His
Ala Asn Tyr Cys Glu Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp
His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val
Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala65 70 75 80 Asn Leu
Lys Ser Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met 85 90 95
Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp 100
105 110 Met Val Val Glu Gly Cys Gly Cys Arg 115 120
145363DNAArtificial SequenceAB2-034 145atgcaagcca aacacaaaca
gcggaagcgt cttaagtcca gctgcaaaag gcaccctttg 60tatgtggact tcagtgatgt
ggggtggaat gactggatca ttgctccctc tggctatcat 120gccaactact
gcgacggaga atgccctttt cctctggctg atcatctgaa ctccactaat
180catgccattg ttcagacgtt ggtcaaccac taccgcatgc ggggccatag
cccctttgcc 240aacctcaaat catgctgtgt cccgaccaag ctgagaccca
tgtccatgtt gtactatgat 300gagaatgaaa aggttgtatt aaagaactat
caggacatgg ttgtggaggg ttgcgggtgt 360cgt 363146121PRTArtificial
SequenceAB2-034 146Met Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys
Ser Ser Cys Lys1 5 10 15 Arg His Pro Leu Tyr Val Asp Phe Ser Asp
Val Gly Trp Asn Asp Trp 20 25 30 Ile Ile Ala Pro Ser Gly Tyr His
Ala Asn Tyr Cys Asp Gly Glu Cys 35 40 45 Pro Phe Pro Leu Ala Asp
His Leu Asn Ser Thr Asn His Ala Ile Val 50 55 60 Gln Thr Leu Val
Asn His Tyr Arg Met Arg Gly His Ser Pro Phe Ala65 70 75 80 Asn Leu
Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met 85 90 95
Leu Tyr Tyr Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp 100
105 110 Met Val Val Glu Gly Cys Gly Cys Arg 115 120
147348DNAArtificial SequenceBMP-2ma 147atggctcaag ccaaacacaa
acagcggaaa cgccttaagt ccagctgtaa gagacaccct 60ttgtacgtgg acttcagtga
cgtggggtgg aatgactgga ttgtggctcc cccggggtat 120cacgcctttt
actgccacgg agaatgccct tttcctctgg ctgatcatct gaactccact
180aatcatgcca ttgttcagac gttggtcaac tctgttaact ctaagattcc
taaggcatgc 240tgtgtcccga cagaactcag tgctatctcg atgctgtacc
ttgacgagaa tgaaaaggtt 300gtattaaaga actatcagga catggttgtg
gagggttgtg ggtgtcgc 348148116PRTArtificial SequenceBMP-2ma 148Met
Ala Gln Ala Lys His Lys Gln Arg Lys Arg Leu Lys Ser Ser Cys1 5 10
15 Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp
20 25 30 Trp Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His
Gly Glu 35 40 45 Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr
Asn His Ala Ile 50 55 60 Val Gln Thr Leu Val Asn Ser Val Asn Ser
Lys Ile Pro Lys Ala Cys65 70 75 80 Cys Val Pro Thr Glu Leu Ser Ala
Ile Ser Met Leu Tyr Leu Asp Glu 85 90 95 Asn Glu Lys Val Val Leu
Lys Asn Tyr Gln Asp Met Val Val Glu Gly 100 105 110 Cys Gly Cys Arg
115 149399DNAArtificial SequenceBMP2/BMP6 149atgcaacaga gtcgtaatcg
ctctacccag tcccaggacg tggcgcgggt ctccagtgct 60tcagattaca acagcagtga
attgaaaaca gcctgcagga agcatgagct gtatgtgagt 120ttccaagacc
tgggatggca ggactggatc attgcaccca agggctatgc tgccaattac
180tgtgatggag aatgctcctt cccactcaac gcacacatga atgcaaccaa
ccacgcgatt 240gtgcagacct tggttcacct tatgaacccc gagtatgtcc
ccaaaccgtg ctgtgcgcca 300actaagctaa atgccatctc ggttctttac
tttgatgaca actccaatgt cattctgaaa 360aaatacagga atatggttgt
aagagcttgt ggatgccac 399150133PRTArtificial SequenceBMP2/BMP6
150Met Gln Gln Ser Arg Asn Arg Ser Thr Gln Ser Gln Asp Val Ala Arg1
5 10 15 Val Ser Ser Ala Ser Asp Tyr Asn Ser Ser Glu Leu Lys Thr Ala
Cys 20 25 30 Arg Lys His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly
Trp Gln Asp 35 40 45 Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn
Tyr Cys Asp Gly Glu 50 55 60 Cys Ser Phe Pro Leu Asn Ala His Met
Asn Ala Thr Asn His Ala Ile65 70 75 80 Val Gln Thr Leu Val His Leu
Met Asn Pro Glu Tyr Val Pro Lys Pro 85 90 95 Cys Cys Ala Pro Thr
Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp 100 105 110 Asp Asn Ser
Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg 115 120 125 Ala
Cys Gly Cys His 130
* * * * *