U.S. patent application number 11/471893 was filed with the patent office on 2006-10-19 for bmp-2 variants with improved properties.
This patent application is currently assigned to Xencor, Inc.. Invention is credited to John R. Desjarlais, Shannon Alicia Marshall, Jonathan Zalevsky.
Application Number | 20060235204 11/471893 |
Document ID | / |
Family ID | 34965017 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060235204 |
Kind Code |
A1 |
Desjarlais; John R. ; et
al. |
October 19, 2006 |
BMP-2 variants with improved properties
Abstract
The invention relates to variants of BMP-2 with improved
properties and methods for their use.
Inventors: |
Desjarlais; John R.;
(Pasadena, CA) ; Marshall; Shannon Alicia; (San
Francisco, CA) ; Zalevsky; Jonathan; (Riverside,
CA) |
Correspondence
Address: |
DORSEY & WHITNEY LLP
555 CALIFORNIA STREET, SUITE 1000
SUITE 1000
SAN FRANCISCO
CA
94104
US
|
Assignee: |
Xencor, Inc.
Monrovia
CA
|
Family ID: |
34965017 |
Appl. No.: |
11/471893 |
Filed: |
June 20, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11097960 |
Mar 31, 2005 |
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11471893 |
Jun 20, 2006 |
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60558189 |
Mar 31, 2004 |
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60570520 |
May 11, 2004 |
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60578432 |
Jun 9, 2004 |
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60587464 |
Jul 13, 2004 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/51 20130101; A61P 19/00 20180101; A61P 19/08 20180101 |
Class at
Publication: |
530/350 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C07K 14/475 20060101 C07K014/475 |
Claims
1-27. (canceled)
28. A non-naturally occuring variant BMP-2 protein comprising at
least one substitution as compared to an amino acid sequence
comprising SEQ ID NO:1, said substitution selected from the group
consisting of: S12E, S13K, K15D, P18R, P18T, S24N, S24Q, N29D,
N29S, W31A, W31E, W31N, W31Q, P36K, P36Q, P36R, P36S, P36T, H39E,
H39Q, F41D, F41N, E46Q, D53Q, D53T, Q64E, L66E, L66N, L66Q, S69A,
S69D, S69R, S69F, V70E, V70K, V70Q, V70T, N71K, S72D, S85E, A86D,
L90E, L90K, Y91D, Y91E, Y91K, Y91T, N95D, N95S, E96D, E96S, E96N,
V98A, V98D, V98R, V98T, L100A, L100E, L100K, L100Q, Y103D, Q104D,
and E109R.
29. A BMP-2 variant protein of claim 28, wherein a substitution is
K15D.
30. A BMP-2 variant protein of claim 28, wherein a substitution is
selected from the group consisting of F41D and F41N.
31. A BMP-2 variant protein of claim 28, wherein a substitution is
selected from the group consisting of S69A, S69D, S69R, and
S69F.
32. A BMP-2 variant protein of claim 28, wherein a substitution is
selected from the group consisting of N95D and N95S.
33. A nucleic acid encoding the non-naturally occurring variant
BMP-2 protein of claim 28.
34. An expression vector comprising the nucleic acid of claim
33.
35. A host cell comprising the nucleic acid of claim 33.
36. A host cell comprising the expression vector of claim 34.
37. A method of producing a non-naturally occurring BMP-2 protein
comprising culturing the host cell of claim 35 under conditions
suitable for expression of said nucleic acid.
38. The method according to claim 37 further comprising recovering
said BMP-2 protein.
39. A pharmaceutical composition comprising the BMP-2 protein of
claim 28, and a pharmaceutical carrier.
40. A method for treating a BMP-2 responsive disorder comprising
administering the non-naturally occurring BMP-2 protein of claim 28
to a patient in need of said treatment.
41. The method according to claim 40, wherein said BMP-2 responsive
disorder is selected from the group consisting of bone fractures,
bone degeneration and spinal fusion.
Description
[0001] This application claims benefit under 35 U.S.C. .sctn.119(e)
to U.S. Ser. No. 60/558,189 filed on Mar. 31, 2004, entitled
"Cysteine Knot Cytokine Variants with Improved Properties"; U.S.
Ser. No. 60/570,520 filed on May 11, 2004 entitled "Cysteine Knot
Cytokine Variants with Improved Properties"; U.S. Ser. No.
60/578,432 filed on Jun. 9, 2004 entitled "Cysteine Knot Cytokine
Variants with Improved Properties"; and U.S. Ser. No. 60/587,464,
filed on Jul. 13, 2004 entitled "Cysteine Knot Cytokine Variants
with Improved Properties", all of which are expressly incorporated
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to variants of bone morphogenetic
proteins and other cysteine knot growth factors with improved
properties, and to methods of making and using compositions
utilizing these variants.
BACKGROUND OF THE INVENTION
[0003] Bone morphogenetic proteins (BMPs) are a well-known family
of growth factors that contribute to developmental processes such
as pattern formation and tissue specification as well as promoting
wound healing and repair processes in adult tissues. BMPs were
initially isolated by their ability to induce bone and cartilage
formation and are now known to regulate cell proliferation,
migration, differentiation, and apoptosis in a number of tissues
and organs.
[0004] BMPs include a number of related human proteins, such as
BMP-2, BMP-3 (osteogenin), BMP-3b (GDF-10), BMP-4 (BMP-2b), BMP-5,
BMP-6, BMP-7 (osteogenic protein-1 or OP-1), BMP-8 (OP-2), BMP-8B
(OP-3), BMP-9 (GDF-2), BMP-10, BMP-11 (GDF-11), BMP-12 (GDF-7),
BMP-13 (GDF-6, CDMP-2), BMP-15 (GDF-9), BMP-16, GDF-1, GDF-3, GDF-5
(CDMP-1), and GDF-8 (myostatin). BMPs may be grouped into
subfamilies. For example, BMP-2 and BMP-4 are closely related, as
are BMP-5, BMP-6, BMP-7, BMP-8, and BMP-8B. BMP-13, BMP-14, and
BMP-12 also constitute a subfamily. BMPs are also present in other
animal species. Furthermore, there is some allelic variation in BMP
sequences among different members of the human population.
[0005] BMPs are a subset of the transforming growth factor-.beta.
(TGF-.beta.) family, which also includes TGFs (TGF-.beta.1,
TGF-.beta.2, and TGF-.beta.3), activins (activin A) and inhibins,
macrophage inhibitory cytokine-1 (MIC-1), Mullerian inhibiting
substance, anti-Mullerian hormone, and glial cell line derived
neurotrophic factor (GDNF). The TGF-.beta. family is in turn a
subset of the cysteine knot cytokine superfamily. Additional
members of the cysteine knot cytokine superfamily include, but are
not limited to, platelet derived growth factor (PDGF), vascular
endothelial growth factor (VEGF), placenta growth factor (PIGF),
noggin, neurotrophins (BDNF, NT3, NT4, and .beta.NGF),
gonadotropin, follitropin, lutropin, interleukin-17, and
coagulogen.
[0006] BMPs have demonstrated utility in the treatment of a variety
of conditions and diseases. BMP-2 and BMP-7 have been used to
promote bone formation, bone fracture healing, and spinal fusion.
BMP-4 (Rundle et. al. (2003) Bone 32: 591-601), BMP-5 (Arosarena
and Collins (2003) Arch. Otolaryngol. Head Neck Surg. 129:
1125-1130), BMP-6 (Helm (2003) Gene Ther. 10: 1735-1743), and BMP-9
(Li et. al. (2003) J. Gene Med. 5: 748-756), have also been
demonstrated to promote bone healing in animal models. Animal
studies indicate that BMP-7 may be used to treat renal fibrosis and
renal failure (Wang et. al. (2001) J. Am. Soc. Nephrol. 12:
2392-2399; Wang and Hirshberg (2003) Am. J. Physiol. Renal Physiol.
284: 1006-1013; Zeisberg et. al. (2003) Nat. Med. 9: 964-968; and
Zeisberg et. al. (2003) Am. J. Physiol. Renal Physiol. 285:
F1060-F1067), ischemic stroke (Chang et. al. (2003) Stroke 34:
558-564 and Harvey et. al. Pharmacol. Ther. (2005) 105: 113-125)
and inflammatory bowel diseases (Maric et. al. (2003) J. Cell
Physiol. 196: 258-264).
[0007] Structurally, BMPs are dimeric cysteine knot proteins. Each
BMP monomer comprises multiple intramolecular disulfide bonds. An
additional intermolecular disulfide bond mediates dimerization in
most BMPs. BMPs may form homodimers; furthermore some BMPs may form
heterodimers. BMPs are expressed as pro-proteins comprising a long
pro-domain, one or more cleavage sites, and a mature domain. The
pro-domain is believed to aid in the correct folding and processing
of BMPs. Furthermore, in some but not all BMPs, the pro-domain may
noncovalently bind the mature domain and may act as an inhibitor
(eg. Thies et. al. (2001) Growth Factors 18: 251-259).
[0008] BMP signal transduction is initiated when a BMP dimer binds
two type I and two type 11 serine/threonine kinase receptors. Type
I receptors include but are not limited to ALK-1, ALK-2 (also
called ActRIa or ActRI), ALK-3 (also called BMPRIa), and ALK-6
(also called BMPRIb) and type II receptors include but are not
limited to ActRIIa (also called ActRII), ActRIIb, and BMPRII.
Following BMP binding, the type II receptors phosphorylate the type
I receptors, the type I receptors phosphorylate members of the Smad
family of transcription factors, and the Smads translocate to the
nucleus and activate the expression of a number of genes.
[0009] BMPs also interact with inhibitors, soluble receptors, and
decoy receptors, including BAMBI (BMP and activin membrane bound
inhibitor), BMPER (BMP-binding endothelial cell precursor-derived
regulator), Cerberus, cordin, cordin-like, Dan, Dante, follistatin,
follistatin-related protein (FSRP), ectodin, gremlin, noggin,
protein related to Dan and cerberus (PRDC), sclerostin,
sclerostin-like, and uterine sensitization-associated gene-1
(USAG-1). Furthermore, BMPs may interact with co-receptors, for
example BMP-2 and BMP-4 bind the co-receptor DRAGON (Samad et. al.
(2005) J. Biol. Chem.), and extracellular matrix components such as
heparin sulfate and heparin (Irie et. al. (2003) Biochem. Biophys.
Res. Commun. 308: 858-865)
[0010] For further background on the BMP family, see Balemans and
Hul (2002) Dev. Biol. 250: 231-250; Bubnoff and Cho (2001) Dev.
Biol. 239: 1-14; Celeste et. al. (1990) Proc. Nat. Acad. Sci. USA
87: 9843-9847; and Cheng et. al. (2003) J. Bone Joint Surgery 85A:
1544-1552
[0011] A number of unfavorable properties of naturally occurring
BMPs limit the development and use of BMP therapeutics. BMP
expression yields are typically poor and suitable expression hosts
are limited, hindering development and production. BMPs often
possess multiple biological effects, including unwanted side
effects. Many BMPs are poorly soluble, reducing storage stability
and bioavailability. Finally, BMPs may induce unwanted immune
responses.
[0012] Earlier studies have identified BMP variants with a number
of interesting properties. BMP variants with improved yield in the
context of E. coli expression and subsequent refolding from
inclusion bodies have been disclosed (U.S. Pat. No. 5,399,677; U.S.
Pat. No. 5,804,416; and U.S. Pat. No. 6,677,432). Consensus BMP
variants with BMP-like activity have also been described (U.S. Pat.
No. 5,011,691; U.S. Pat. No. 6,395,883; U.S. Pat. No. 6,531,445). A
BMP-2 point mutant, L51P, has been described that does not bind
type I receptors but binds type II receptors normally (Keller et.
al. (2004) Nat. Struct. Mol. Biol. 11: 481-488). Deletion mutants
of BMP-4 that act as competitive inhibitors of BMP signaling have
been disclosed (Weber et. al. (2003) J. Bone Miner. Res. 18:
2142-2151). Mutagenesis experiments have also been performed on
BMP-2 to identify residues important for receptor binding; some of
these variants were found to act as antagonists (Kirsch et. al.
(2000) EMBO J. 19: 3314-3324 and Nickel et. al. (2001) J. Bone
Joint Surg. Am. 83-A: S7-S14). In addition, methods for identifying
analogs of morphogenetic proteins have been claimed (U.S. Pat. No.
6,273,598). Furthermore, several point mutants of ActA with reduced
ALK-4 binding have been identified: S60P, 163P, M91 E, 1105E, and
M108A (Harrison et. al. (2004) J. Biol. Chem. 279:
28036-28044).
SUMMARY OF THE INVENTION
[0013] The present invention is related to variants of human bone
morphogenetic proteins and other cysteine knot cytokine proteins
with improved properties, including increased expression yield,
expression in the absence of a pro-domain, increased solubility,
increased specific activity, altered receptor, co-receptor, and
inhibitor specificity, and decreased immunogenicity.
[0014] In one aspect, the invention provides variant BMP-7 protein
comprising the sequence:
Fx(1-20)-Vb(21)-Fx(22-38)-Vb(39)-Fx(40-64)-Vb(65)-Fx(66-71)-Vb(72)-Fx(73--
77)-Vb(78)-Fx(79-92)-Vb(93)-Fx(94-119)-Vb(120)-Fx(121-134)-Vb(135)
[0015] wherein [0016] Fx1(1-20) corresponds to amino acid residues
1-20 of human BMP-7 (SEQ ID NO:5); [0017] Vb(21) is selected from
the group consisting of L and G; [0018] Fx(22-38) corresponds to
amino acid residues 22-38 of human BMP-7 (SEQ ID NO:5); [0019]
Vb(39) is selected from the group consisting of K, A and S; [0020]
Fx(40-64) corresponds to amino acid residues 40-64 of human BMP-7
(SEQ ID NO:5); [0021] Vb(65) is selected from the group consisting
of Y and N; [0022] Fx(66-71) corresponds to amino acid residues
66-71 of human BMP-7 (SEQ ID NO:5); [0023] Vb(72) is selected from
the group consisting of A and D; [0024] Fx(73-77) corresponds to
amino acid residues 73-77 of human BMP-7 (SEQ ID NO:5); [0025]
Vb(78) is selected from the group consisting of Y and H; [0026]
Fx(79-92) corresponds to amino acid residues 79-92 of human BMP-7
(SEQ ID NO:5); [0027] Vb(93) is selected from the group consisting
of F, H, S and T; [0028] Fx(94-119) corresponds to amino acid
residues 94-119 of human BMP-7 (SEQ ID NO:5); [0029] Vb(120) is
selected from the group consisting of S and D; [0030] Fx(121-134)
corresponds to amino acid residues 121-134 of human BMP-7 (SEQ ID
NO:5); [0031] Vb(135) is selected from the group consisting of A
and E; [0032] wherein the variant comprises an amino acid
substitution as compared to human BMP-7 (SEQ ID NO:5).
[0033] In a further aspect, the invention provides variant BMP-7
proteins comprising a substitution as compared to human BMP-7 (SEQ
ID NO:5) selected from the group consisting of: L21G, K39A, K39S,
Y65N, A72D, Y78H, F93H, F93S, F93T, SI 20D and A135E. In both
cases, particular variants with single substitutions include, L21G,
K39A, K39S, Y65N, A72D, Y78H, F93H, F93S, F93T, S120D and A135E.
Particular sets of substitutions include, K39S-F93S; K39S-S120D;
K39S-S120D-Y65N; K39S-S120D-A72D; K39S-S120D-Y78H; K39S-S120D-F93H;
K39S-S120D-F93S; Y65N-L21G; Y65N-L21R; Y65N-K39S; Y65N-Y78H;
Y65N-S120D; Y78H-A72D; Y78H-F93H-Y65N; Y78H-F93H-A72D;
Y78H-F93H-S120D; Y78H-S120D and F93H-K39S.
[0034] In an additional aspect, the invention provides variant
BMP-7 proteins having altered receptor binding affinity compared to
wild-type BMP-7 (SEQ ID NO:5), the variant BMP-7 protein comprising
one or more substitutions selected from the group consisting of:
M23N, Q53G, Q53H and I86D.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows sequence alignments of the mature domains of
human BMP and GDF proteins (Residues 14-114 of SEQ ID NO:1,
residues 16-116 of SEQ ID NO:2, residues 37-138 of SEQ ID NO:3,
residues 38-139 of SEQ ID NO:4, residues 38-139 of SEQ ID NO:5,
residues 38-139 of SEQ ID NO:6 and SEQ ID NOS:71-83). The consensus
BMP sequence and the MIC-1 sequence are shown for reference. The
N-terminal most residues of the mature domain, which are
significantly less well aligned, are not shown.
[0036] FIG. 2 shows the hexameric structure comprising BMP-7 dimer
bound to two type I receptors and two type II receptors. The
structure was generated by superimposing BMP-2 (white) bound to
ALK-3 (gray, upper right and lower left) and BMP-7 (black) bound to
ActRIIa (gray, upper left and lower right).
[0037] FIG. 3 shows alignments of human type I BMP receptors and
human type II BMP receptors used for homology modeling (SEQ ID
NOS:7-12).
[0038] FIG. 4 shows 12-point binding curves for wild-type BMP-7
(Image clone) binding to ActRIIa, BMPRII, BMPRIa, and BMPRIb.
[0039] FIG. 5 shows 12-point binding curves for (A) BMPRIa (ALK-3),
(B) BMPRIb (ALK-6), (C) ActRIIa, and (D) BMPRII. The thick black
lines are wild type BMP-7 (Image clone). The thin gray lines
correspond to variants.
[0040] FIG. 6 shows dose-response C2C12 biassay data for selected
Library 1 variants. Highlighted variants in (A) include: F93Q
(black hollow circles), F93S (gray filled diamonds), N110D (gray
filled squares), S120D (black hollow squares), A135E (black hollow
triangles), A135S (black filled circles) and wild type (thick black
line, no markers). Additional variants are shown in (A) and (B)
[0041] FIG. 7 shows 12-point binding curves for (A) BMPRIa (ALK-3),
(B) BMPRIb (ALK-6), (C) ActRIIa, and (D) BMPRII. Two replicates of
wild type (Image clone) are shown (thick black line, filled black
circles). Variants are shown in thin gray lines, no markers.
[0042] FIG. 8 shows a summary of 12-point receptor binding curves
for wild type human BMP-7 (Image clone) and variants.
[0043] FIG. 9 shows dose-response C2C12 biassay data for selected
Library 2 variants.
[0044] FIG. 10 shows dose-response C2C12 biassay data for selected
Library 3 variants, Library 1 variants (thick gray lines, no
markers), and wild type (thick black line, no markers).
[0045] FIG. 11 shows (A) ELISA quantitation of the expression yield
of the best single, double, and triple variants in 293T cells
(black bars) and CHO cells (white bars) and (B) that enhanced
expression yield of the best-expressing single, double, and triple
mutant variants results in increased C2C12 bioactivity from
serially diluted conditioned media.
[0046] FIG. 12 shows appropriate correction factors when using a
commercial ELISA (R&D Systems) to determine the concentration
of selected BMP-7 variants.
[0047] FIG. 13 shows purification of selected BMP-7 variants.
[0048] FIG. 14 shows fluorescence images of SDS-PAGE gels showing
(A) Alexa 568 labeling of BMP-7 variant Y65N/S120D as a function of
dye concentration; and (B) scale-up of Alexa-568 labeled Y65N/S120D
BMP-7.
[0049] FIG. 15 shows fluorescence anisotropy as a function of
receptor concentration.
[0050] FIG. 16 shows that binding of Alexa568-labeled Y65N/S120D
BMP-7 to the receptor ActRIIa can be competed with unlabeled
BMP-7.
[0051] FIG. 17 shows competitive binding of (A) recombinant human
BMP-7 (R&D Systems), (B) BMP-7 variant 565 (Y65N/F93T/R129D),
(C) BMP-7 variant 526 (K39S/S120D/R134E), and (D) BMP-7 variant 504
(Y65N/S120D) to the BMP receptors and inhibitors BMPRIb (open
circles), ActRIIa (closed diamonds), BMPRII (closed triangles), and
Noggin (stars) determined using AlphaScreen.
[0052] FIG. 18 shows a bar graph indicating the EC50 of binding for
four BMP-7 variants to three receptors and one inhibitor.
DETAILED DESCRIPTION OF THE INVENTION
[0053] By "BMP responsive disorders" and grammatical equivalents
herein is meant diseases, disorders, and conditions that may
benefit from treatment with one or more BMPs. Examples of BMP
responsive disorders include, but are not limited to, cartilage,
bone, and tooth disorders or conditions including but not limited
to bone fractures, bone degeneration, osteoporosis, spinal fusion,
spinal degenerative disc disease, osteotomy, orthopedic and
reconstructive surgery, and periodontal disease; repair of tendons
and ligaments; renal disease including but not limited to chronic
or acute renal failure, renal injury due to reperfusion,
drug-induced renal toxicity, renal fibrosis, renal osteodystrophy,
and vascular complications resulting from kidney disease; liver
disease including but not limited to cirrhosis and hepatic
fibrosis; lung disease including but not limited to asthma,
emphysema, and pulmonary fibrosis; wound healing; cancers including
but not limited to prostate cancer; inflammatory bowel disease;
conditions that would benefit from a neuroprotective agent
including but not limited to stroke, Parkinson's disease, traumatic
brain injury, and amyotrophic lateral sclerosis; and skin and hair
disorders. By "exposed residues" and grammatical equivalents herein
are meant those residues whose side chains have at least 50
.ANG..sup.2 (square Angstroms) of solvent accessible surface area
in the context of a specified protein structure, preferably an
x-ray crystal structure. As will be appreciated by those skilled in
the art, other values such as 75 .ANG..sup.2 (square Angstroms) or
fractional values such as 50% could be used instead. Furthermore,
alternative methods such as contact models, among others, may be
used to identify exposed residues. By "expression yield" and
grammatical equivalents herein is meant the amount of protein,
preferably in mg/L or PCD (picograms per cell per day) that is
produced or secreted under a given expression protocol (that is, a
specific expression host, transfection method, media, time, etc.).
By "improved expression yield" and grammatical equivalents herein
is meant an increase in expression yield, relative to a wild type
or parent protein, under a given set of expression conditions. In a
preferred embodiment, at least a 50% improvement is achieved, with
improvements of at least 100%, 5-fold, 10-fold, or more being
especially preferred. In another preferred embodiment, the
expression yield is improved to yields of at least 1 .mu.g/ml, with
at least 10 .mu.g/ml or 100 .mu.g/ml being especially preferred. By
"hydrophobic residues" and grammatical equivalents are meant
valine, isoleucine, leucine, methionine, phenylalanine, tyrosine,
and tryptophan. By "interface residues" and grammatical equivalents
herein are meant those residues located within 8 .ANG. (Angstroms)
of a protein-protein contact. Distances of less than 5 .ANG.
(Angstroms) are especially preferred. Distances may be measured in
the context of any structure, with high-resolution crystal
structures being especially preferred. By "library" as used herein
is meant a collection of protein sequences that are likely to take
on a particular fold or have particular protein properties. The
library preferably comprises a set of sequences resulting from
computation, which may include energy calculations or statistical
or knowledge based approaches. Libraries that range in size from
about 5 to about 10.sup.13 sequences are preferred. Libraries are
generally generated experimentally and analyzed for the presence of
members possessing desired protein properties. By "mature domain"
herein is meant, in the context of BMP-7, a domain substantially
comprising residues 1-137 of BMP-7. In wild type BMP-7, the mature
domain is cleaved from the pro-domain by furin or a furin-like
proprotein convertase. By "modification" and grammatical
equivalents is meant one or more insertions, deletions, or
substitutions to a protein or nucleic acid sequence. By "naturally
occurring" or "wild type" or "wt" and grammatical equivalents
thereof herein is meant an amino acid sequence or a nucleotide
sequence that is found in nature, including allelic variations. In
a preferred embodiment, the wild type sequence is the most
prevalent human sequence. However, the wild type BMP nucleic acids
and proteins may be a less prevalent human allele or BMP nucleic
acids and proteins from any number of organisms, including but not
limited to rodents (rats, mice, hamsters, guinea pigs, etc.),
primates, and farm animals (including sheep, goats, pigs, cows,
horses, etc). By "nucleic acid" and grammatical equivalents herein
is meant DNA, RNA, or molecules which contain both deoxy- and
ribonucleotides. Nucleic acids include genomic DNA, cDNA and
oligonucleotides including sense and anti-sense nucleic acids.
Nucleic acids may also contain modifications, such as modifications
in the ribose-phosphate backbone that confer increased stability
and half-life. By "polar residues" and grammatical equivalents
herein are meant aspartic acid, asparagine, glutamic acid,
glutamine, lysine, arginine, histidine, serine, and threonine. By
"pro-domain" herein is meant, in the context of a BMP or other
TGF-.beta. family member, the N-terminal domain that is removed
following cleavage by furin or a furin-like proprotein convertase.
The presence of the pro-domain may promote proper folding and
processing. By "protein" herein is meant a molecule comprising at
least two covalently attached amino acids, which includes proteins,
polypeptides, oligopeptides and peptides. The protein may be made
up of naturally occurring amino acids and peptide bonds, or
synthetic peptidomimetic structures such as peptoids (see Simon et
al. (1992) Proc. Natl. Acad. Sci. USA 89: 9367-9371). For example,
homo-phenylalanine, citrulline, and noreleucine are considered
amino acids for the purposes of the invention, and both D- and
L-amino acids may be utilized. By "protein properties" herein are
meant physical, chemical, and biological properties including but
not limited to physical properties (including molecular weight,
hydrodynamic properties such as radius of gyration, net charge,
isoelectric point, and spectral properties such as extinction
coefficient), structural properties (including secondary, tertiary,
and quaternary structural elements) stability (including thermal
stability, stability as a function of pH or solution conditions,
storage stability, and resistance or susceptibility to
ubiquitination, proteolytic degradation, or chemical modifications
such as methionine oxidation, asparagine and glutamine deamidation,
sidechain racemerization or epimerization, and hydrolysis of
peptide bonds), solubility (including susceptibility to aggregation
under various conditions, oligomerization state, and
crystallizability), kinetic and dynamic properties (including
flexibility, rigidity, folding rate, folding mechanism, allostery,
and the ability to undergo conformational changes and correlated
motions), binding affinity and specificity (to one or more
molecules including proteins, nucleic acids, polysaccharides,
lipids, and small molecules, and including affinities and
association and dissociation rates), enzymatic activity (including
substrate specificity; association, reaction, and dissociation
rates; reaction mechanism; and pH profile), ammenability to
synthetic modification (including PEGylation and attachment to
other molecules or surfaces), expression properties (such as yield
in one or more expression hosts, soluble versus inclusion body
expression, subcellular localization, ability to be secreted, and
ability to be displayed on the surface of a cell), processing and
posttranslational modifications (including proteolytic processing,
N- or C-linked glycosylation, lipidation, sulfation, and
phosphorylation), pharmacokinetic and pharmacodynamic properties
(including bioavailability following subcutaneous, intramuscular,
oral, or pulmonary delivery; serum half-life, distribution, and
mechanism and rate of elimination) and ability to induce altered
phenotype or changed physiology (including immunogenicity,
toxicity, ability to signal or inhibit signaling, ability to
stimulate or inhibit cell proliferation, differentiation, or
migration, ability to induce apoptosis, and ability to treat
disease). By "solubility" and grammatical equivalents herein is
meant the maximum possible concentration of protein, in the desired
or physiologically appropriate oligomerization state, in a solution
of specified condition (i.e. pH, temperature, concentration of any
buffer components, salts, detergents, osmolytes, etc.). Unless
otherwise noted, dimeric BMPs are the desired species. By "soluble
expression" and grammatical equivalents herein is meant that the
protein is able to be produced at least partially in soluble form
rather than in inclusion bodies when expressed in a prokaryotic
host. It is preferred that at least 1 .mu.g soluble protein is
produced per 100 mL culture, with at least 10 .mu.g or 100 .mu.g
being especially preferred. By "improved solubility" and
grammatical equivalents herein is meant an increase in the maximum
possible concentration of protein, in the desired or
physiologically appropriate oligomerization state, in solution. For
example, if the naturally occurring protein can be concentrated to
1 mM and the variant can be concentrated to 5 mM under the same
solution conditions, the variant can be said to have improved
solubility. In a preferred embodiment, solubility is increased by
at least a factor of 2, with increases of at least 5-fold or
10-fold being especially preferred. As will be appreciated by those
skilled in the art, solubility is a function of solution
conditions. For the purposes of this invention, solubility should
be assessed under solution conditions that are pharmaceutically
acceptable. Specifically, pH should be between 6.0 and 8.0, salt
concentration should be between 50 and 250 mM. Additional buffer
components such as excipients may also be included; although it is
preferred that albumin is not required. By "variant BMP nucleic
acids" and grammatical equivalents herein is meant nucleic acids
that encode variant BMPs. Due to the degeneracy of the genetic
code, an extremely large number of nucleic acids may be made, all
of which encode the variant BMPs of the present invention, by
simply modifying the sequence of one or more codons in a way that
does not change the amino acid sequence of the variant BMP. By
"variant BMPs" or "non-naturally occurring BMPs" and grammatical
equivalents thereof herein is meant non-naturally occurring BMPs
which differ from a wild type or parent BMP by at least one (1)
amino acid insertion, deletion, or substitution. It should be noted
that unless otherwise stated, all positional numbering of variant
BMPs and variant BMP nucleic acids is based on these sequences. BMP
variants are characterized by the predetermined nature of the
variation, a feature that sets them apart from naturally occurring
allelic or interspecies variation of the BMP sequence. BMP variants
must retain at least 50% of wild type BMP activity in one or more
cell types, as determined using an appropriate assay described
below. Variants that retain at least 75% or 90% of wild type
activity are more preferred, and variants that are more active than
wild type are especially preferred. Alternatively, in some
embodiments BMP variants may be engineered to have different
activities than a wild type BMP. For example, competitive
inhibitors may be designed. A variant BMP may contain insertions,
deletions, and/or substitutions at the N-terminus, C-terminus, or
internally. In a preferred embodiment, variant BMPs have at least 1
residue that differs from the most similar human BMP sequence, with
at least 2, 3, 4, or 5 different residues being more preferred.
Variant BMPs may contain further modifications, for instance
mutations that alter additional protein properties such as
stability or immunogenicity or which enable or prevent
posttranslational modifications such as PEGylation or
glycosylation. Variant BMPs may be subjected to co- or
post-translational modifications, including but not limited to
synthetic derivatization of one or more side chains or termini,
glycosylation, PEGylation, circular permutation, cyclization,
fusion to proteins or protein domains, and addition of peptide tags
or labels.
[0054] Naturally occurring BMPs regulate cell proliferation,
migration, differentiation, and apoptosis in a number of tissues
and organs; as a result BMPs may serve many therapeutic uses.
However, naturally occurring BMPs are difficult to produce in large
amounts, are sparingly soluble, exhibit pleiotropic activities, and
may induce unwanted immune responses.
[0055] Here, are disclosed novel variants of human BMPs. These BMP
variants comprise one or more modifications that were selected to
improve biophysical properties and clinical performance.
[0056] Strategies for Improving Expression Yield
[0057] Reported expression yields for BMPs are typically very low.
Reported yields range from 2-6 ng/mL for transiently transfected
COS-1 cells in roller bottle culture to 100-200 ng/mL in
DHFR-amplified stably transfected CHO cells, see U.S. Pat. No.
6,048,964 to John C. Lee, et al. To facilitate the development and
therapeutic use of BMPs, it would be desirable to increase the
expression yield to at least 10 .mu.g/ml, with at least 100
.mu.g/ml being more preferred and at least 1000 .mu.g/ml being
especially preferred.
[0058] A number of nucleic acid properties and protein properties
may influence expression yields; furthermore the expression host
and expression protocol contribute to yields. Any of these
parameters may be optimized to improve expression yields. Also,
expression yield may be improved by the incorporation of one or
mutations that confer improved stability and/or solubility, as
discussed further below. Furthermore, interactions between the
pro-domain and the mature domain may influence folding efficiency,
and so the pro-domain may also be targeted for modification.
[0059] In a preferred embodiment, nucleic acid properties are
optimized to improve expression yields using one or more of the
following strategies: 1) replace imperfect Kozak sequence, 2)
reduce 5' GC content and secondary structure of the RNA, 3)
optimize codon usage, 4) use an alternate leader sequence, 5)
include a chimeric intron, or 6) add an optimized poly-A tail to
the C-terminus of the message. In another preferred embodiment,
protein properties are optimized to improve expression yields using
one or more of the following strategies: 1) optimize the signal
sequence, 2) optimize the proteolytic processing site, 3) replace
one or more cysteine residues in order to minimize formation of
improper disulfide bonds, 4) improve the rate or efficiency of
protein folding, or 5) increase protein stability, especially
proteolytic stability. In an alternate preferred embodiment,
alternate pro-domain sequences are used. For example, the
pro-domain from BMP-2 may be used to aid in the expression of BMP-4
(Wozney et. al. (1988) Science 242: 1528-1534). Pro-domains that
may be used include but are not limited to the pro-domains from any
BMP sequence and the MIC-1 pro-domain. The pro-domain may be
expressed in cis or in trans.
[0060] In an additional preferred embodiment, transfection or
expression conditions are optimized to increase expression yields.
For example, since furin and other pro-protein convertase enzymes
require calcium, the addition of calcium to the media during
expression may increase the yield of properly processed protein. As
another example, proteosome inhibitors may be added to minimize
proteosomal degradation. Fetal calf serum or heparin may also be
used. In a further preferred embodiment, the expression host is
selected to optimize expression yields. Folding and processing of
BMPs is relatively complex and may be assisted by appropriate
chaperones. These chaperones may not be expressed equally in all
mammalian cell lines. BMP-7 is naturally produced in the kidney and
several well-established expression lines are derived from the
kidney; in a preferred embodiment BMP-7 is expressed in a kidney
cell line including but not limited to 293T, 239-EBNA, COS, and
BHK. In another preferred embodiment, the cleavage site in BMP-7 is
optimized to promote more efficient proteolytic processing by furin
and related subtilisin-like proprotein convertase enzymes.
Substrate preferences for furin have been well-characterized
(Henrich et. al. (2003) Nat. Struct. Biol. 10: 520-526; Holyoak et.
al. (2004) Biochem. 43: 2412-2421; and Duckert et. al. (2004) PEDS
17: 107-112), and cleavage sites in BMP and TGF-.beta. proteins
have been analyzed (Constam and Robertson (1999) JBC 144: 139-149).
In an alternate preferred embodiment, BMP-7 is expressed in a cell
line that is co-transfected with one or more chaperone or
processing proteins, including but not limited to furin.
[0061] Strategies for Enabling the Use of Alternate Expression
Hosts
[0062] BMPs are typically expressed in mammalian cells. In order to
enable the use of alternate expression systems, including but not
limited to yeast expression systems, it would be desirable to 1)
eliminate the N-linked glycosylation site, 2) eliminate potential
O-linked glycosylation sites, 3) enable expression in the absence
of the pro-domain, and 4) enable processing by an alternate
protease present in the desired expression host. In a preferred
embodiment, one or more N- or O-linked glycosylation sites is
removed. Removal of glycosylation sites from variant BMP
polypeptides may be accomplished, for example, by the elimination
of one or more serine or threonine residues to the native sequence
or variant BMP polypeptide (for O-linked glycosylation sites) or by
the modification of a canonical N-linked glycosylation site,
N-X-Y-X, where X is any amino acid except for proline and Y is
threonine, serine or cysteine. In another preferred embodiment,
pro-domain dependence is reduced or eliminated by 1) introducing
mutations that stabilize the folded state of the BMP; 2) reducing
the exposed hydrophobic surface area of BMP; 3) stabilizing one or
more intermediates along the folding pathway of BMP; or 4)
replacing one or more pairs of cysteine residues forming a
disulfide bond. In an additional preferred embodiment, the furin
cleavage site is modified to allow recognition by an alternate
protease that is present in the desired expression host. For
example, the furin cleavage site may be changed to a kexin cleavage
site to facilitate yeast expression. Kexin cleavage sites have been
well characterized; see for example Holyoak et. al. (2004) Biochem.
43: 2412-2421.
[0063] In an alternate preferred embodiment, the BMPs are expressed
using in vitro translation. A number of factors may be added to the
reaction to improve the yield of total protein and of correctly
folded protein, including but not limited to 1) pro-domains from
any TGF-.beta. family member, including but not limited to BMP-2,
BMP-4, BMP-7, and MIC-1; 2) accessory factors and chaperones
including but not limited to cysteine isomerases, proline
isomerases, BiP, heat shock proteins, furin, and other proprotein
convertases; 3) redox agents including but not limited to
glutathione; 4) monovalent and divalent cations including but not
limited to sodium, potassium, calcium, zinc, and magnesium; and 5)
microsomes.
[0064] Strategies for Improving Solubility
[0065] A variety of strategies may be utilized to design BMP
variants with improved solubility and expression yield. In a
preferred embodiment, one or more of the following strategies are
used: 1) reduce hydrophobicity by substituting one or more
solvent-exposed hydrophobic residues with suitable polar residues,
2) increase polar character by substituting one or more neutral
polar residues with charged polar residues, 3) increase protein
stability, for example by one or more modifications that improve
packing in the hydrophobic core, increase beta sheet forming
propensity, improve helix capping and dipole interactions, or
remove unfavorable electrostatic interactions (increasing the
stability of a protein may improve solubility by decreasing the
population of partially folded or misfolded states that are prone
to aggregation), and 4) modify one or more residues that can affect
the isoelectric point of the protein (that is, aspartic acid,
glutamic acid, histidine, lysine, arginine, tyrosine, and cysteine
residues). Protein solubility is typically at a minimum when the
isoelectric point of the protein is equal to the pH of the
surrounding solution. Modifications that perturb the isoelectric
point of the protein away from the pH of a relevant environment,
such as serum, may therefore serve to improve solubility.
Furthermore, modifications that decrease the isoelectric point of a
protein may improve injection site absorption (Holash et. al.
(2002) Proc. Nat. Acad. Sci. USA 99: 11393-11398).
[0066] Strategies for Altering Receptor Binding Affinity or
Specificity
[0067] Several strategies may be used to design BMP variants with
improved receptor binding affinity or specificity. In a preferred
embodiment, diversity is incorporated at one or more receptor
interface positions. However, as is known in the art, modifications
at positions distal to the receptor binding interface may also
alter binding affinity or specificity. In an especially preferred
embodiment, modifications are made to positions in BMP that contact
one or more non-conserved receptor positions. For example, Arg 48,
Gln 53, and Glu 60 in BMP-7 contacts position 76 in the type II
receptor; this position is Glu in ActRIIb, Lys in ActRII, and Thr
in BMPRII. Some BMPs, such as BMP-2, bind more tightly to type I
receptors while other BMPs, such as BMP-7, have higher affinity for
type II receptors. In an additional preferred embodiment, a BMP is
modified such that its lower affinity receptor binding site is made
more similar to a BMP for which that site is the higher affinity
site. For example, the type I receptor interface of BMP-7 may be
modified to be more similar to BMP-2, or the type II receptor
interface of BMP-2 may be altered to be more BMP-7-like. In an
alternate embodiment, modifications are made to stabilize the bound
conformation of BMP versus the free conformation. For example,
BMP-7 binds first to the type II receptor and then to the type I
receptor, and binding to the type II receptor may increase binding
affinity for the type I receptor. Binding to the type II receptor
causes a conformational change in BMP, producing what may be a
higher affinity state. Accordingly, mutations may be introduced to
stabilize the bound state.
[0068] Strategies for Evading BMP Inhibitors
[0069] A number of soluble and membrane bound proteins function as
endogenous inhibitors of BMP action. In a preferred embodiment,
BMPs are engineered to reduce or eliminate binding affinity for one
or more BMP inhibitors while retaining affinity for one or more of
the BMP receptors. Rational alteration of inhibitor specificity may
be used to control the site of BMP action, as many of the
inhibitors are expressed in specific tissues or organs. Similar
approaches may be used to alter specificity for co-receptors such
as DRAGON, extracellular matrix components such as heparin and
heparin sulfate, and serum components such as
alpha2-macroglobulin.
[0070] Protein Design and Engineering Methods
[0071] A number of methods can be used to identify modifications
(that is, insertion, deletion, or substitution mutations) that will
yield BMP variants with improved properties. These methods include,
but are not limited to, sequence profiling (Bowie and Eisenberg
(1991) Science 253: 164-170), rotamer library selections (Dahiyat
and Mayo (1996) Protein Sci 5: 895-903; Dahiyat and Mayo, Science
(1997) 278: 82-87; Desjarlais and Handel (1995) Prot. Sci. (1995)
4: 2006-2018; Harbury et. al. (1995) Proc. Nat. Acad. Sci. USA 92:
8408-8412; Kono et al., Proteins (1994) 19: 244-255; Hellinga and
Richards (1994) Proc. Nat. Acad. Sci. USA 91: 5803-5807); and
residue pair potentials (Jones (1994) Prot. Sci. 3: 567-574).
[0072] In a preferred embodiment, one or more sequence alignments
of BMPs and related proteins is analyzed to identify residues that
are likely to be compatible with each position. In a preferred
embodiment, the PFAM or BLAST alignment algorithm is used to
generate alignments of the BMP subfamily, the TGF-.beta. family, or
the cysteine knot cytokine superfamily. For each variable position,
suitable substitutions may be defined as those residues that are
observed at the same position in homologous sequences. Especially
preferred substitutions are those substitutions that are frequently
observed in homologous sequences. In an additional preferred
embodiment, an Analogous Contact Environment (ACE) algorithm, U.S.
patent application Ser. No. 11/00,647, filed Dec. 8, 2004, is used
in conjunction with the sequence alignment information to identify
alternate suitable residues that are located in structurally
similar environments in other BMPs or homologs. In an especially
preferred embodiment, rational design of improved BMP variants is
achieved by using Protein Design Automation.RTM. (PDA.RTM.)
technology; see U.S. Pat. Nos. 6,188,965; 6,269,312; 6,403,312;
6,708,120; WO98/47089 and U.S. Ser. Nos. 09/058,459, 09/127,926,
60/104,612, 60/158,700, 09/419,351, 60/181,630, 60/186,904,
09/419,351, 09/782,004 and 09/927,790, 60/347,772, and 10/218,102;
and PCT/US01/218,102 and U.S. Ser. No. 10/218,102, U.S. Ser. No.
60/345,805; U.S. Ser. No. 60/373,453 and U.S. Ser. No. 60/374,035,
or using the sequence prediction algorithm (SPA) (Raha et al.
(2000) Protein Sci., 9: 1106-1119; U.S. Ser. No. 09/877,695, filed
Jun. 8, 2001 and 10/071,859, filed Feb. 6, 2002).
[0073] Structural Analysis of BMPS
[0074] Obtaining Structures of BMPS
[0075] PDA.RTM. technology calculations require a template protein
structure. In one embodiment, the structure of a human BMP is
obtained by x-ray crystallography or NMR. Structures of BMPs
include BMP-2 (PDB code 3BMP, Scheufler et. al. (1999) J. Mol.
Biol. 287: 103), BMP-2 mutant L51P (PDB code 1 REU, Keller et. al.
(2004) Nat. Struct. Mol. Biol. 11: 481) and wild type human BMP-7
(PDB code 1 LXI Griffith et. al. (1996) Proc. Nat. Acad. Sci. USA
93: 878-883). It is also possible to use the crystal structure of
another cysteine knot cytokine protein, such as TGF-.beta.2 (PDB
code 1TFG, Schlunegger and Grutter (1992) Nature 358: 430), or NMR
structures of cysteine knot cytokine proteins such as TFG-.beta.1
(PDB codes 1KLA, 1KLC, and 1KLD; Hinck et. al. (1996) Biochem. 35:
5817) In an especially preferred embodiment, the crystal structure
is a co-crystal structure comprising a BMP and a BMP receptor.
High-resolution structures are available for BMP-7 in complex with
the receptor ActRIIa (PDB code 1LX5, Greenwald et. al. (2003) Mol.
Cell 11: 605-617), activin A bound to ActRIIb (PDB codes 1NYS and
1NYU, Thompson et. al. (2003) EMBO J. 22: 1555-1566), and BMP-2
bound to ALK-3 (PDB code 1ES7, Kirsch et. al. (2000) Nat. Struct.
Biol. 7: 492; and PDB code 1REW, Keller et. al. (2004) Nat. Struct.
Mol. Biol. 11: 481). In another preferred embodiment, the crystal
structure is a co-crystal structure comprising BMP and a BMP
inhibitor. A high resolution structure is available for BMP-7 bound
to the soluble inhibitor noggin (PDB code 1M4U, Groppe et. al.
(2002) Nature 420: 636). Structures of additional BMPs alone and
bound to one or more receptors or inhibitors may be built using NMR
or x-ray crystal structures including but not limited to those
described above in conjunction with homology modeling, structural
alignment, and protein-protein docking methods known in the
art.
[0076] Identifying Furin Cleavage Sites
[0077] The furin cleavage sites of BMPs may be determined by
scanning for the consensus furin cleavage site, R-X-X-K/R (SEQ ID
NO:84), in the residues located N-terminally relative to the
aligned regions of the BMP mature domains. The sequence of BMP-7 in
the region of the cleavage site is (from P8 to P1 before the
cleavage site and from P1' to P4' after the cleavage site, with "|"
indicating the cleaved bond) is EVHLRSIR|STGG (SEQ ID NO:85),
wherein the residues "STGG" comprise residues 1 through 4 of the
BMP-7 mature domain (SEQ ID NO:5). The most favored furin cleavage
site comprises R at P4, R at P1, and K or R at P2. It is also
favorable to have at least one basic residue in residues P5-P8.
Some BMPs have multiple cleavage sites. For example, BMP-4 has two
cleavage sites, and sequential cleavage is thought to provide a
mechanism for regulation of activation and signaling range (Degnin
et. al. (2004) Mol. Biol. Cell 15: 5012-5020).
[0078] Identifying Glycosylation Sites
[0079] BMP-7 has an N-linked glycosylation site at Asn 80. When
expressed in mammalian cells, the mature domain of BMP-7 does not
include any O-linked glycosylation sites. However, it is possible
that one or more serine or threonine residues would be susceptible
to glycosylation in alternate expression hosts, including but not
limited to yeast and Baculovirus expression systems. The presence
and location of such O-linked glycosylation sites may be determined
experimentally, for example using mass spectrometry.
[0080] Identifying Solvent-Exposed Hydrophobic Residues
[0081] As used herein, exposed hydrophobic residues in BMP-7
include but are not limited to Tyr 44, Trp 52, Trp 55, Ile 57, Phe
73, Tyr 78, Ile 86, Leu 90, Phe 93, Ile 94, Leu 115, Tyr 116, Tyr
117, Val 123, Leu 125, and Tyr 128. BMP-7 also contains three
hydrophobic residues in the disordered N-terminal region (that is,
residues 1-35). While these residues are not observed in the
crystal structures of BMP-7, it is highly likely that they are
significantly exposed to solvent. In a preferred embodiment, these
additional hydrophobic residues, Leu 21, Met 23, and Val 26, are
also considered solvent exposed hydrophobic residues.
[0082] Identifying Residues at the Receptor Binding Sites
[0083] In a preferred embodiment, residues that mediate
intermolecular interactions between BMPs and their receptors are
replaced with structurally and functionally compatible residues
that confer improved receptor binding affinity or specificity.
Preferred residues at the BMP/type I receptor interface include,
but are not limited to, residues Lys 39, Phe 47, Asp 49, Leu 50,
Gly 51, Pro 74, Leu 75, Asn 76, Ser 77, Tyr 78, Asn 80, Asn 83, Ile
86, Leu 90, Phe 93, Ile 94, Pro 96, Tyr 116, Lys 126, Tyr 128, Arg
129, Asn 130, and Met 131. Preferred residues at the BMP/type II
receptor interface include, but are not limited to, residues Tyr
44, Arg 48, Gln 53, Ile 57, Ala 58, Pro 59, Glu 60, Gly 61, Tyr 62,
Ala 63, Gln 108, Asn 110, Ala 111, Ile 112, Ser 113, Val 114, Leu
115, Phe 117, Asn 122, Val 123, Leu 125, Lys 127, and Arg 134.
[0084] Identifying Residues at Inhibitor Binding Sites
[0085] In a preferred embodiment, residues that mediate
intermolecular interactions between BMPs and their inhibitors are
replaced with structurally and functionally compatible residues
that confer reduced inhibitor binding affinity or increased
inhibitor binding specificity. Preferred residues at the
noggin/BMP-7 interface include, but are not limited to, Phe 73, Pro
74, Leu 75, Asn 76, Ser 77, Asn 83, Ile 86, Val 87, and Leu 90.
[0086] Identifying Residues in Regions of High Electrostatic
Potential
[0087] Proteins may be destabilized by the presence of unfavorable
electrostatic interactions or stabilized by the presence of
favorable electrostatic interactions. Accordingly, a protein may be
stabilized by removing unfavorable electrostatic interactions or by
incorporating favorable electrostatic interactions. Modifying
regions of high electrostatic potential may also modulate
interactions with serum and extracellular matrix components, which
may affect pharmacokinetics properties. In a preferred embodiment,
the electrostatic potential that is present at each residue
position is determined, for example by using Debye-Huckel
calculations. Residues in BMP-7 that are located in regions of
electrostatic potential greater than 0.25 or less than -0.25
include, but are not limited to, Lys 40, Ser 46, Arg 48, Tyr 62,
Ala 64, Tyr 65, Tyr 66, Cys 67, Glu 68, Gly 69, Glu 70, Cys 71, Ala
72, Tyr 78, Asn 80, Ala 81, Thr 82, Asn 83, His 84, Ala 85, Val 87,
Gln 88, Thr 89, Ile 94, Pro 100, Cys 104, Ala 105, Pro 106, Thr
107, Gin 108, Leu 109, Asn 110, Ala 111, Ile 112, Ser 113, Asn 122,
Ile 124, Asn 130, Met 131, Val 132, Val 133, Arg 134, Ala 135, Cys
136, Gly 137, and His 139.
[0088] Design of Optimized BMP Variants
[0089] Identifying Suitable Modifications of the Furin Cleavage
Site
[0090] The furin cleavage sequences of several BMPs differ somewhat
from the consensus furin cleavage sequence. In BMP-7, preferred
modifications to improve proteolytic processing include, but are
not limited to, (P8) E.fwdarw.Q or K; (P6) H.fwdarw.K or R; (P5)
L.fwdarw.K; and (P2) I.fwdarw.K or R.
[0091] Identifying Suitable Replacements for Glycosylation
Sites
[0092] In a preferred embodiment, residues comprising a N- or
O-linked glycosylation site are replaced with structurally and
functionally compatible residues that do not comprise a
glycosylation site. As is known in the art, N-linked glycosylation
sites are specified by the sequence N-X-(S/T)-X (SEQ ID NO:86),
where X may be any residue other than proline. Accordingly, an
N-linked glycosylation site may be eliminated by 1) replacing the N
with any other residue, 2) replacing either X with proline, or 3)
replacing the S or T with any residue other than T, S, or C.
Preferred modifications that remove the N-linked glycosylation site
include, but are not limited to, replacing Asn 80 with Asp, Gln,
Ser, or Thr; replacing Thr 82 with Val, and replacing Asn 83 with
Pro.
[0093] Identifying Suitable Polar Residues for Each Exposed
Hydrophobic Position
[0094] In a preferred embodiment, solvent exposed hydrophobic
residues are replaced with structurally and functionally compatible
polar residues. Alanine and glycine may also serve as suitable
replacements, constituting a reduction in hydrophobicity.
Furthermore, mutations that increase polar character, such as Phe
to Tyr, and mutations that reduce hydrophobicity, such as Ile to
Val, may be appropriate. In a preferred embodiment, preferred
suitable polar residues are defined as those polar residues: 1)
whose energy in the optimal rotameric configuration, as determined
using PDA.RTM. technology, is more favorable than the energy of the
exposed hydrophobic residue at that position and 2) whose energy in
the optimal rotameric configuration is among the most favorable of
the set of energies of all polar residues at that position. In a
preferred embodiment, the polar residues that are included in the
library at each variable position are deemed suitable by both
PDA.RTM. technology calculations and by sequence alignment data.
Alternatively, one or more of the polar residues that are included
in the library are deemed suitable by either PDA.RTM. technology
calculations or sequence alignment data.
[0095] Especially preferred modifications to BMP-7 include, but are
not limited to, the following substitutions: L21D, L21G, L21K,
L21N, L21R, L21S, M23D, M23G, M23K, M23N, M23R, M23S, V26D, V26E,
V26G, V26K, V26N, V26S, Y44A, Y44D, Y44E, Y44G, Y44H, Y44K, Y44N,
Y44P, Y44Q, Y44R, Y44S, W52A, W52E, W52K, W52Q, W55A, W55E, W55H,
W55K, W55N, W55Q, I57A, I57D, I57E, I57H, I57K, I57L, I57T, I57V,
E60R, F73A, F73D, F73E, F73H, F73Q, F73R, F73S, Y78D, Y78G, Y78H,
Y78N, Y78R, Y78S, Y78T, I86A, I86D, I86E, I86K, I86Q, I86T, L90E,
L90K, L90N, L90Q, L90R, L90S, L90T, F93A, F93D, F93E, F93G, F93H,
F93Q, F93R, F93S, F93T, I94A, I94E, I94H, I94K, I94Q, I94R, I94T,
L115E, L1115K, L115T, Y116A, Y116D, Y116E, Y116H, Y116K, Y116S,
Y116T, F117A, F117D, F117E, F117H, F117K, F117Q, F117R, F117Y,
V123A, V123D, V123N, V123R, V123T, L125A, L125E, L125K, L125Q,
Y128D, Y128E, Y128H, Y128K, and Y128Q. Most especially preferred
modifications are those modifications that confer improved
properties, such as improved expression yield or activity. Most
especially preferred modifications to exposed hydrophobic residues
in BMP-7 include, but are not limited to, L21G, L21R, M23G, M23N,
M23R, M23S, V26G, V26N, Y65N, Y78H, Y78R, I86A, F93D, F93E, F93G,
F93H, F93S, F93T, 194R, Y116H, F117H, F117Y, and Y128D.
[0096] Identifying Suitable Residues for Each Interface
Position
[0097] Suitable residues for interface residues as used herein are
meant all amino acid residues that are compatible with the
structure of a BMP and that retain appreciable binding affinity for
at least one of the BMP receptors. Alternatively, competitive
inhibitor variants may be generated by identifying alternate
residues that are compatible with the structure of a BMP but that
substantially eliminate binding affinity for at least one of the
BMP receptors. Suitable residues may confer binding specificity by
maintaining or increasing affinity for one or two receptors or
inhibitors while substantially reducing binding affinity for the
other receptors, inhibitors, or additional binding partners. In
other cases, modifications are selected to confer other desired
properties, for example improved expression yield, while
maintaining binding affinities that are substantially similar to
the wild type protein. Typically, the interface positions will be
substantially exposed to solvent. In such cases, preferred
substitutions include the polar residues, alanine, and glycine.
However, for interface positions that are substantially buried in
the dimer structure, hydrophobic replacements are preferred.
Suitable polar residues may also include the subset of polar
residues that are observed in analogous positions in homologous
proteins, especially other BMPs. In an especially preferred
embodiment, suitable polar residues include the subset of polar
residues with low or favorable energies as determined using
PDA.RTM. technology calculations or SPA calculations (described
above).
[0098] Especially preferred modifications to polar BMP-7 interface
residues include, but are not limited to, K39D, K39E, K39G, K39N,
K39R, K39S, K39T, R48D, R48E, R48H, R48K, R48N, R48Q, Q53A, Q53D,
Q53E, Q53G, Q53H, Q53K, Q53R, Q53S, Q53T, E60H, E60K, E60N, E60P,
E60Q, E60R, E60S, E60T, N76A, N76D, N76S, N76T, S77A, S77D, S77E,
S77H, S77K, S77N, S77P, S77Q, S77T, K126D, K126E, K126G, K126Q,
K126R, K127A, K127D, K127E, K127H, K127N, K127P, K127Q, K127S,
K127T, R129D, R129E, R129K, R129N, R129S, R134D, R134E, R134K,
R134Q, and R134S. Most especially preferred modifications are those
modifications that confer improved properties, such as improved
expression yield, improved activity, or enhanced receptor binding
specificity. Most especially preferred modifications to residues in
regions of high electrostatic potential in BMP-7 include, but are
not limited to, K39A, K39S, R48H, R48N, R48Q, Q53A, Q53K, Q53D,
Q53G, Q53S, Q53T, E60R, K126R, K127E, R129D, R129N, R134E, and
R134S. Additional especially preferred modifications are those
modifications that reduce binding to either type I or type II
receptors, thereby potentially acting as a competitive inhibitor of
BMP. Additional especially preferred modifications to receptor
interface residues in BMP-7 include, but are not limited to, Y44T,
W52E, and 157Q.
[0099] Further preferred modifications are those modifications that
alter binding affinity or specificity to a BMP inhibitor. Preferred
modifications that reduce binding to noggin include, but are not
limited to, W55I, W55L, W55K, W55R, I57M, I57Y, I57E, I57H, I57K,
I57Q, I57R, A58I, A58L, A58M, A58Y, A58V, A58E, A58H, A58K, A58Q,
A58R, P59Y, N76E, N76Q, N76R, S77E, S77Q, N83F, N83W, N83Y, N83H,
N83K, N83R, I86L, I86M, I86F, I86Y, I86R, V87H, S113I, S113L,
S113M, S113F, S113Y, S113E, S113H, S113K, S113Q, S113R, L115M,
L115K, L115R, V123M, V123Y, V123H, K1271, K127V, K127H, Y1281, and
Y128R. Preferred modifications that increase binding to noggin
include, but are not limited to, R48M, P59M, E601, E60L, E60M,
E60V, P74M, N76I, N76V, N76A, S77T, D119I, D119L, K126W, and
K127M.
[0100] Identifying Suitable Residues for Regions of High
Electrostatic Potential
[0101] Regions of high electrostatic potential may be modified in
order to increase protein stability or to alter receptor binding
affinity and specificity. In a preferred embodiment, residues that
are located in a region of high electrostatic potential are
replaced by structurally and functionally compatible residues that
are predicted to interact favorably with the local electrostatic
field. In a preferred embodiment, suitable polar residues include
the subset of electrostatically favorable residues that are
observed in analogous positions in homologous proteins, especially
other BMPs. In an especially preferred embodiment, suitable polar
residues include the subset of polar residues with low or favorable
energies as determined using PDA.RTM. technology calculations or
SPA calculations (described above).
[0102] Especially preferred modifications to BMP-7 residues located
in regions of high electrostatic potential include, but are not
limited to, Q88E, N110D, N110E, N110H, A111D, A111S, N130D, A135D,
A135E, and A135S. Most especially preferred modifications are those
modifications that confer improved properties, such as improved
expression yield, improved activity, or enhanced receptor binding
specificity. Most especially preferred modifications to receptor
interface residues in BMP-7 include, but are not limited to, N110D,
A135E, and A135S.
[0103] Identifying Suitable Residues for Additional Surface
Positions
[0104] Additional residues on the surface of a BMP may be modified
in order to improve stability, solubility, or expression yield. In
a preferred embodiment, suitable polar residues include the subset
of polar residues that are observed in analogous positions in
homologous proteins, especially other BMPs. In an especially
preferred embodiment, suitable polar residues include the subset of
polar residues with low or favorable energies as determined using
PDA.RTM. technology calculations or SPA calculations (described
above).
[0105] Additional especially preferred modifications to BMP-7
surface residues include but are not limited to Q36E, Q36N, Q36R,
E42D, E42Q, E42R, E42T, D49E, D49S, D54K, D54N, D54R, D54S, E70A,
E70Q, N95D, N95K, N95Q, N95R, E97D, E97K, E97R, T98A, T98E, T98K,
T98R, Q108D, Q108K, Q108S, D119E, D119N, D119S, D119T, S120D,
S120E, S120N, S120R, S121D, S121E, S121K, S121N, S121T, N122E,
N122Q, and N122R. Furthermore, residue T98 may be deleted. Most
especially preferred modifications are those modifications that
confer improved properties, such as improved expression yield,
improved activity, or enhanced receptor binding specificity. Most
especially preferred modifications to additional residues in BMP-7
include, but are not limited to, S120D.
[0106] Identifying Suitable Combinations of Mutations
[0107] In a preferred embodiment, variants comprising two or more
mutations, including but not limited to those disclosed above, are
made. Such variants may exhibit greater improvements in expression
yield, solubility, or receptor specificity than point mutants. Such
variants may also exhibit improvements in more than one protein
property.
[0108] Especially preferred variants comprising two mutations
include but are not limited to L21G/F93H, L21R/F93H, M23N/Y65N,
M23R/Y65N, K39A/Y65N, K39A/F93H, K39S/Y78H, K39S/F93H, K39S/N110D,
K39S/S120D, K39S/N130D, K39S/R134E, K39S/A135E, R48N/F93H,
Q53D/Y65N, Q53G/Y65N, Q53G/Y78H, Q53S/Y65N, Q53T/Y65N, 157L/Y65N,
Y65N/Y78H, Y65N/Y78R, Y65N/S120D, Y65N/A135E, Y65N/A135S,
A72D/F93H, Y78H/F93H, Y78H/A105V, Y78H/Q108D, Y78H/Y116H,
Y78H/F117Y, Y78H/S120D, Y78H/N130D, Y78H/R134E, Y78H/R134S,
Y78H/A135E, Y78H/A135S, Y78H/H139R, Y78R/F93H, F93H/F117Y,
F93H/S120D, F93H/R134S, and F93H/H139R. Especially preferred
variants comprising three mutations include but are not limited to
L21 G/K39S/S120D, M23R/K39S/S120D, K39S/Y65N/S120D,
K39S/A72D/S120D, K39S/Y78H/S120D, K39S/Q108D/S120D, Y65N/Y78H/F93H,
Y65N/Y78H/R134E, A72D/Y78H/F93H, Y78H/F93H/Q108D, Y78H/F93H/F117H,
Y78H/F93H/S120D, and Y78H/F93H/R134E. Especially preferred variants
comprising four modifications include but are not limited to
K39S/F93S/Q108D/S120D, K39S/F93S/S120D/R129D, K39S/Y65N/F93S/S120D,
K39S/Y78H/F93S/S120D, K39S/F93S/S120D/R134E, K39S/A72D/F92S/S120D,
Y65N/Y78H/F93S/R134E, A72D/Y78H/F93S/R134E, M23R/Y65N/F93S/R129D,
Y65N/F93S/Q108D/R129D, K39S/F93T/Q108D/S120D,
K39S/F93T/S120D/R129D, K39S/Y65N/F93T/S120D, K39S/Y78H/F93T/S120D,
K39S/F93T/S120D/R134E, K39S/A72D/F93T/S120D, Y65N/Y78H/F93T/R134E,
A72D/Y78H/F93T/R134E, M23R/Y65N/F93T/R129D, and
Y65N/F93T/Q108D/R129D.
[0109] Additional Modifications
[0110] Additional modifications that might favorably impact
expression yield and/or activity can be deduced by observing
significant trends in the data obtained. Once such trend is the
observation that introduction of a negatively charged amino (E or
D) within the Finger 2 region of BMP7 (positions 105-139) leads in
most cases to enhanced expression or activity, exemplified by the
expression and activity of variants such as Q108D, N110D, N110E,
S120D, K127E, Y128D, R129D, N130D, and A135E. Analysis of
additional positions within this region indicates that BMP7
substitutions T107D, T107E, S113D, S113E will most likely also
possess superior expression and/or activity. A second trend is that
the substitution of exposed hydrophobic amino acids with more polar
or less hydrophobic alternatives generally leads to enhanced
expression yield or activity, exemplified by variants such as Y78H,
186A, Y128D, and multiple substitutions of F93. Application of this
trend to I124, another exposed hydrophobic residue, suggests the
additional expression- or activity-enhancing variants I124A, I124D,
I124E, I124K, I124N, I124Q, I124R, I124S, I124T, and I124V.
[0111] Additional insertions, deletions, and substitutions may be
incorporated into the variant BMPs of the invention in order to
confer other desired properties. In a preferred embodiment, the BMP
variant comprises insertions, deletions, or substitutions that
reduce immunogenicity, as described in "Antibodies And Fc Fusion
Proteins With Altered Immunogenicity," U.S. Ser. No. 60/643,313,
filed Jan. 12, 2005. In an alternate preferred embodiment, the BMP
variant is further modified to increase stability. As discussed
above, modifications that improve stability can also improve
solubility, for example by decreasing the concentration of
partially unfolded, aggregation-prone species. For example,
modifications can be introduced to the protein core that improve
packing or remove polar or charged groups that are not forming
favorable hydrogen bond or electrostatic interactions. It is also
possible to introduce modifications that introduce stabilizing
electrostatic interactions or remove destabilizing interactions.
Additional stabilizing modifications also may be used. In another
preferred embodiment, one or more cysteine, lysine, histidine, or
other reactive amino acids are added to or eliminated from variant
BMPs in order to incorporate or remove sites that are susceptible
to covalent modification. For example, see "Rational Chemical
Modification," U.S. patent application Ser. No. 10/956,352, filed
Sep. 30, 2004. As is known in the art, variant BMPs may be modified
by adding an epitope tag (e.g. a poly-histidine (poly-His), c-myc,
or FLAG-tag) or a fusion partner (e.g. an immunoglobulin, the Fc
region of an immunoglobulin, albumin, other BMPs, other cytokine
proteins, the extracellular domain of a BMP receptor protein, etc).
For further details see the descriptions of tags and fusion
partners in "Optimized Fc Variants," U.S. Patent Application No.
60/627,774, filed Nov. 12, 2004.
[0112] BMP Forms
[0113] BMPs are naturally expressed as pro-proteins comprising a
long pro-domain, one or more cleavage sites, and a mature domain.
This pro-protein is then processed by the cellular machinery to
yield a dimeric mature BMP molecule. In a preferred embodiment, the
variants of the invention are produced in a similar manner. The
pro-domain is believed to aid in the correct folding and processing
of BMPs. Furthermore, in some but not all BMPs, the pro-domain may
noncovalently bind the mature domain and may act as a chaperone, as
well as an inhibitor (eg. Thies et. al. (2001) Growth Factors 18:
251-259). In additional preferred embodiments, the variants of the
invention are produced and/or administered therapeutically in this
form. In alternative embodiments, BMPs may be produced in other
forms, including, but not limited to, mature domain produced
directly or refolded from inclusion bodies, or full-length intact
pro-protein. The variants of the invention are expected to find use
in these and other forms.
[0114] Generating The Variants
[0115] Variant BMP nucleic acids and proteins of the invention may
be produced using a number of methods known in the art, as
elaborated upon below.
[0116] Preparing Nucleic Acids Encoding the BMP Variants
[0117] In a preferred embodiment, nucleic acids encoding BMP
variants are prepared by total gene synthesis, or by site-directed
mutagenesis of a nucleic acid encoding wild type or variant BMP.
Methods including template-directed ligation, recursive PCR,
cassette mutagenesis, site-directed mutagenesis or other techniques
that are well known in the art may be utilized (see for example
Strizhov et. al. PNAS 93:15012-15017 (1996), Prodromou and Perl,
Prot. Eng. 5: 827-829 (1992), Jayaraman and Puccini, Biotechniques
12: 392-398 (1992), and Chalmers et. at. Biotechniques 30: 249-252
(2001)).
[0118] Expression Vectors
[0119] In a preferred embodiment, an expression vector that
comprises the components described below and a gene encoding a
variant BMP is prepared. Numerous types of appropriate expression
vectors and suitable regulatory sequences for a variety of host
cells are known in the art. The expression vectors may contain
transcriptional and translational regulatory sequences including
but not limited to promoter sequences, ribosomal binding sites,
transcriptional start and stop sequences, translational start and
stop sequences, transcription terminator signals, polyadenylation
signals, and enhancer or activator sequences. In a preferred
embodiment, the regulatory sequences include a promoter and
transcriptional start and stop sequences. In addition, the
expression vector may comprise additional elements. For example,
the expression vector may have two replication systems, thus
allowing it to be maintained in two organisms, for example in
mammalian or insect cells for expression and in a prokaryotic host
for cloning and amplification. Furthermore, for integrating
expression vectors, the expression vector contains at least one
sequence homologous to the host cell genome, and preferably two
homologous sequences, which flank the expression construct. The
integrating vector may be directed to a specific locus in the host
cell by selecting the appropriate homologous sequence for inclusion
in the vector. Constructs for integrating vectors are well known in
the art. In addition, in a preferred embodiment, the expression
vector contains a selectable marker gene to allow the selection of
transformed host cells. Selection genes are well known in the art
and will vary with the host cell used. The expression vectors may
be either self-replicating extrachromosomal vectors or vectors
which integrate into a host genome.
[0120] The expression vector may include a secretory leader
sequence or signal peptide sequence that provides for secretion of
the variant BMP from the host cell. Suitable secretory leader
sequences that lead to the secretion of a protein are known in the
art. The signal sequence typically encodes a signal peptide
comprised of hydrophobic amino acids, which direct the secretion of
the protein from the cell. The protein is either secreted into the
growth media or, for prokaryotes, into the periplasmic space,
located between the inner and outer membrane of the cell. For
expression in bacteria, bacterial secretory leader sequences,
operably linked to a variant BMP encoding nucleic acid, are usually
preferred.
[0121] Transfection/Transformation
[0122] The variant BMP nucleic acids are introduced into the cells
either alone or in combination with an expression vector in a
manner suitable for subsequent expression of the nucleic acid. The
method of introduction is largely dictated by the targeted cell
type. Exemplary methods include CaPO.sub.4 precipitation, liposome
fusion (eg. using the reagent Lipofectin.RTM. or FuGene),
electroporation, viral infection (eg. as outlined in
PCT/US97/01019), dextran-mediated transfection, polybrene mediated
transfection, protoplast fusion, direct microinjection, etc. The
variant BMP nucleic acids may stably integrate into the genome of
the host cell or may exist either transiently or stably in the
cytoplasm.
[0123] Hosts for the Expression of BMP Variants
[0124] Appropriate host cells for the expression of BMP variants
include yeast, bacteria, archaebacteria, fungi, and insect and
animal cells, including mammalian cells. Of particular interest are
fungi such as Saccharomyces cerevisiae and Pichia pastoris and
mammalian cell lines including 293 (eg. 293-T and 293-EBNA), BRK,
CHO (eg. CHOK1 and DG44), COS, Jurkat, NIH3T3, etc. (see the ATCC
cell line catalog). BMP variants can also be produced in more
complex organisms, including but not limited to plants (such as
corn, tobacco, and algae) and animals (such as chickens, goats,
cows); see for example Dove, Nature Biotechnol. 20: 777-779 (2002).
In one embodiment, the cells may be additionally genetically
engineered, that is, contain exogenous nucleic acid other than the
expression vector comprising the variant BMP nucleic acid.
[0125] Expression Methods
[0126] The variant BMPs of the present invention are produced by
culturing a host cell transformed with an expression vector
containing nucleic acid encoding a variant BMP, under the
appropriate conditions to induce or cause expression of the variant
BMP. Either transient or stable transfection methods may be used.
The conditions appropriate for variant BMP expression will vary
with the choice of the expression vector and the host cell, and
will be easily ascertained by one skilled in the art through
routine experimentation.
[0127] Purification
[0128] In a preferred embodiment, the BMP variants are purified or
isolated after expression. Standard purification methods include
electrophoretic, molecular, immunological and chromatographic
techniques, including ion exchange, hydrophobic, affinity, and
reverse-phase HPLC chromatography, and chromatofocusing. For
example, a BMP variant may be purified using a standard
anti-recombinant protein antibody column. Ultrafiltration and
diafiltration techniques, in conjunction with protein
concentration, are also useful. For general guidance in suitable
purification techniques, see Scopes, R., Protein Purification,
Springer-Verlag, NY, 3d ed. (1994). The degree of purification
necessary will vary depending on the desired use, and in some
instances no purification will be necessary.
[0129] Posttranslational Modification and Derivatization
[0130] Once made, the variant BMP may be covalently modified.
Covalent and non-covalent modifications of the protein are thus
included within the scope of the present invention. Such
modifications may be introduced into a variant BMP polypeptide by
reacting targeted amino acid residues of the polypeptide with an
organic derivatizing agent that is capable of reacting with
selected side chains or terminal residues. Optimal sites for
modification can be chosen using a variety of criteria, including
but not limited to, visual inspection, structural analysis,
sequence analysis and molecular simulation. Sites for modification
may be located in the pro-domain or the mature domain.
[0131] In one embodiment, the variant BMP of the invention are
labeled with at least one element, isotope or chemical compound. In
general, labels fall into three classes: a) isotopic labels, which
may be radioactive or heavy isotopes; b) immune labels, which may
be antibodies or antigens; and c) colored or fluorescent dyes. The
labels may be incorporated into the compound at any position.
Labels include but are not limited to biotin, tag (e.g. FLAG, Myc)
and fluorescent labels (e.g. fluorescein). Derivatization with
bifunctional agents is useful, for instance, for cross linking a
variant BMP to a water-insoluble support matrix or surface for use
in the method for purifying anti-variant BMP antibodies or
screening assays, as is more fully described below. Commonly used
cross linking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate. Other modifications
include deamidation of glutaminyl and asparaginyl residues to the
corresponding glutamyl and aspartyl residues, respectively,
hydroxylation of proline and lysine, phosphorylation of hydroxyl
groups of seryl or threonyl residues, methylation of the amino
groups of lysine, arginine, and histidine side chains (T. E.
Creighton, Proteins: Structure and Molecular Properties, W.H.
Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of
the N-terminal amine, and amidation of any C-terminal carboxyl
group. Such derivatization may improve the solubility, absorption,
transport across the blood brain barrier, serum half-life, and the
like. Modifications of variant BMP polypeptides may alternatively
eliminate or attenuate any possible undesirable side effect of the
protein. Moieties capable of mediating such effects are disclosed,
for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack
Publishing Co., Easton, Pa. (1980).
[0132] Another type of covalent modification of variant BMP
comprises linking the variant BMP polypeptide to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol ("PEG"),
polypropylene glycol, or polyoxyalkylenes, in the manner set forth
in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337. A variety of coupling chemistries may be
used to achieve PEG attachment, as is well known in the art.
Examples, include but are not limited to, the technologies of
Shearwater and Enzon, which allow modification at cysteine residues
and primary amines, including but not limited to histidine groups,
lysine groups and the N-terminus (see, Kinstler et al, Advanced
Drug Deliveries Reviews, 54, 477-485 (2002) and M J Roberts et al,
Advanced Drug Delivery Reviews, 54, 459-476 (2002)). Both labile
and non-labile PEG linkages may be used. An additional form of
covalent modification includes coupling of the variant BMP
polypeptide with one or more molecules of a polymer comprised of a
lipophililic and a hydrophilic moiety. Such composition may enhance
resistance to hydrolytic or enzymatic degradation of the BMP.
Polymers utilized may incorporate, for example, fatty acids for the
lipophilic moiety and linear polyalkylene glycols for the
hydrophilic moiety. The polymers may additionally incorporate
acceptable sugar moieties as well as spacers used for BMP
attachment. Polymer compositions and methods for covalent
conjugation are described, for example, in U.S. Pat. Nos.
5,681,811; 5,359,030.
[0133] Another type of modification is chemical or enzymatic
coupling of glycosides to the variant BMP. Such methods are
described in the art, e.g., in WO 87/05330 published 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981). Alternatively, removal of carbohydrate moieties present on
the variant BMP polypeptide may be accomplished chemically or
enzymatically. Chemical deglycosylation techniques are known in the
art and described, for instance, by Hakimuddin, et al., Arch.
Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal.
Biochem., 118:131 (1981). 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., Meth.
Enzymol., 138:350 (1987).
[0134] Assaying the Expression Yield of the Variants
[0135] A primary object of the current invention is the
identification of variant BMPs with increased expression yield.
Accordingly, the yield, using one or more set of expression
conditions, of the variant and wild type BMPs is determined. In one
embodiment, expression yields are determined using ELISA. One
limitation of this technique is that some variants may confer
increased or decreased antibody binding affinity. Accordingly, in a
preferred embodiment ELISAs are performed using at least two
monoclonal antibodies that recognize distinct epitopes. It is also
possible to derive ELISA correction factors for selected variants
by purifying said variants and determining their concentration
through orthogonal methods, such as UV absorption or BCA assay.
Alternatively, the BMPs may be engineered to contain a tag, such as
a FLAG tag or His tag, and anti-tag antibodies may be used in the
ELISA. In another embodiment, expression yields are determined
using Western blotting. As with ELISA, a limitation is that some
mutations may confer increased or decreased antibody binding
affinity.
[0136] Assaying the Solubility of the Variants
[0137] In a preferred embodiment, the variant BMPs are assayed for
solubility using methods including but not limited to those
described below. In all preferred embodiments, the variant and wild
type proteins are compared directly in the same assay system and
under the same conditions in order to evaluate the solubility of
each variant. The solubility of the BMP variants may be determined
under a number of solution conditions. A variety of excipients,
including solubilizing and stabilizing agents, may be tested for
their ability to promote the highest soluble BMP concentration. In
addition, different salt concentrations and varying pH may be
tested. In a preferred embodiment, solubility is assayed under
pharmaceutically acceptable conditions.
[0138] Differential light scattering (DLS) may be used to determine
oligomerization state. DLS determines diffusion coefficients based
on signal correlation from fluctuation of laser light scattered
from Brownian motion of particles in solution (Heimenz, Chapter 10
in Polymer Chemistry, Marcel Dekker, Inc., NY, 1984, pp. 659-701).
Commercially available instruments provide graphical or table
readouts of particle population(s) by size(s) after transforming
the diffusion coefficient(s) measured by
deconvolution/autocorrelation of laser light scattering data using
the Stokes-Einstein equation. The size is therefore the
hydrodynamic radius. The distribution of particle sizes within a
population(s) is the dispersity, and this factor provides data on
the uniformity of the particle population(s). Both dispersity and
the appearance of aggregates over time may be monitored to test for
solubility. Aggregated protein may be easily resolved by DLS, and
readily detected at low levels due to the physical property of
aggregates: they scatter more laser light per unit due to the
greater target surface area. The sample may be directly introduced
into the cuvette (i.e. it is not necessary to perform a
chromatographic step first). A relative ratio of monodisperse to
aggregate particle population may be determined. Optionally, this
ratio may be weighted by mass or by light scattering intensity.
Thus, DLS is a preferred technique to monitor formation of
aggregates, and holds the advantage in that it is a non-intrusive
technique.
[0139] In another preferred embodiment analytical
ultracentrifugation (AUC) is used to determine the oligomerization
state of the variant BMPs. AUC can be performed in two different
`modes`, either velocity or equilibrium. Equilibrium AUC is the
most preferred method for determining protein molecular weight and
oligomeric state measurement.
[0140] A further preferred embodiment is to use size-exclusion
chromatography (SEC) to determine the oligomerization state of the
BMP variants. Utilizing high performance liquid chromatography,
sample may be introduced to an isocratic mobile phase and separated
on a gel permeation matrix designed to exclude protein on the basis
of size. Thus, the samples will be "sieved" such that the
aggregated protein will elute first with the shortest retention
time, and will be easily separated from the remainder. This can
identify aggregates and allow a relative quantification by peak
integration using the peak analysis software provided with the
instrument.
[0141] In an alternate embodiment, protein concentration is
monitored as a function of time. In the case of poor solubility,
aggregates will form over time in the protein solution, and
eventually precipitate entirely. This may be performed following
centrifugation and sampling of the solution phase, in which case
insolubility can be measured as a drop in solution protein
concentration over time will be observed following
centrifugation.
[0142] In an alternate embodiment, the oligomerization state is
determined by monitoring relative mobility on native gel
electrophoresis.
[0143] In another embodiment, the amount of protein that is
expressed solubly is determined. While factors other than the
solubility of the native protein can impact levels of soluble
expression, improvements in soluble expression may correlate with
improvements in solubility. Any of a number of methods may be used;
for example, following expression, SDS-polyacrylamide gel
electrophoresis and/or western blots can be done on the soluble
fraction of crude cell lysates or the expression media.
[0144] Furthermore, any of a number of high throughput screens for
soluble expression may be used. In one embodiment, the protein of
interest is fused to a fluorescent protein such as GFP, and the
cells monitored for fluorescence (Waldo et. al. Nat. Biotechnol.
17: 691 (1999)). In an alternate embodiment, the protein of
interest is fused to the antibiotic resistance enzyme
chloramphenicol transferase. If the protein expresses solubly, the
enzyme will be functional, thereby allowing growth on media with
increased concentration of the antibiotic chloramphenicol (Maxwell
et. al. Protein Sci. 8: 1908 (1999)). In another embodiment, the
protein of interest is expressed as a fusion with the alpha domain
of the enzyme beta-galactosidase. If the protein expresses in
soluble form, the alpha domain will complement the omega domain to
yield a functional enzyme. This may be detected as blue rather than
white colony formation when the cells are plated on media
containing the indicator X-gal (Wigley et. al. Nat. Biotechnol. 19:
131 (2001)).
[0145] Assaying the Activity of the Variants
[0146] In a preferred embodiment, the activity of the wild-type and
variant proteins are analyzed using in vitro receptor binding
assays, cell-based activity assays, or in vivo activity assays.
[0147] Receptor Binding Assays
[0148] In a preferred embodiment, the affinity of the variant BMPs
for one or more BMP receptors is determined. In an especially
preferred embodiment, affinities for ALK-2, ALK-3, ALK-6, ActRII,
ActRIIb, and BMPRII are determined. Suitable binding assays
include, but are not limited to, ELISA, fluorescence anisotropy and
intensity, scintilation proximity assays (SPA) Biacore (Pearce et
al., Biochemistry 38:81-89 (1999)), DELFIA assays, and
AlphaScreen.TM. (commercially available from PerkinElmer; Bosse R.,
Illy C., and Chelsky D (2002)).
[0149] In a preferred embodiment, Biacore or surface plasmon
resonance assays (see for example McDonnell (2001) Curr. Opin.
Chem. Biol. 5:572-577) are used to determine the affinity of one or
more BMP variants for one or more BMP receptors. Biacore
experiments may be performed, for example, by binding BMP
receptor-Fc fusion proteins to a protein A derivitized chip or an
NTA chip and testing each BMP variant as an analyte. It is also
possible to bind an anti-BMP antibody to the chip, or to bind the
BMP variant to the chip and test soluble receptor or Fc-receptor
fusion proteins as analytes. Biacore experiments have been used
previously to characterize binding of TGF-.beta. isoforms to their
receptors (De Crescenzo et. al. (2001) J. Biol. Chem. 276:
29632-29643, De Crescenzo et. al. (2003) J. Mol. Biol. 328:
1173-1183).
[0150] In an alternate preferred embodiment, a plate-based Direct
Binding Assay is used to determine the affinity of one or more BMP
variants for one or more BMP receptors. This method is a modified
sandwich ELISA in which BMP is captured using an anti-BMP
monoclonal antibody and then detected using a BMP receptor/Fc
fusion protein.
[0151] In another preferred embodiment, AlphaScreen.TM. assays
(Bosse R., Illy C., and Chelsky D (2002). Principles of
AlphaScreen.TM. PerkinElmer Literaure Aplication Note Ref# s4069.
http://lifesciences.perkinelmer.com/Notes/S4069-0802.pdf) are used
to characterize receptor and inhibitor binding. AlphaScreen.TM. is
a bead-based non-radioactive luminescent proximity assay where the
donor beads are excited by a laser at 680 nm to release singlet
oxygen. The singlet oxygen diffuses and reacts with the thioxene
derivative on the surface of acceptor beads leading to fluorescence
emission at .about.600 nm. The fluorescence emission occurs only
when the donor and acceptor beads are brought into close proximity
by molecular interactions occurring when each is linked to ligand
and receptor (or ligand and inhibitor) respectively. This
interaction may be competed away by adding an appropriate amount of
unlabeled BMP variant that binds the relevant receptor or
inhibitor.
[0152] In one embodiment, AlphaScreen.TM. assays are performed
using 1) BMP modified by the addition of a suitable tag or label;
2) donor beads capable of binding the tag or label used to modify
the BMP; 3) a BMP receptor or inhibitor modified by the addition of
a suitable tag or label; 4) acceptor beads capable of binding the
tag or label used to modify the BMP receptor, and 5) varying
amounts of an unlabeled variant BMP-7 molecule, which acts as a
competitor. It is also possible to coat the donor or acceptor beads
with antibodies that specifically recognize the native BMP or BMP
receptor, or to bind the receptor to the donor beads and the ligand
to the acceptor beads. In an alternate embodiment, AlphaScreen.TM.
assays are performed using 1) a type I BMP receptor modified by the
addition of a suitable tag or label; 2) donor beads capable of
binding the tag or label used to modify the type I BMP receptor; 3)
a type II BMP receptor modified by the addition of a suitable tag
or label; 4) acceptor beads capable of binding the tag or label
used to modify the type II BMP receptor; 5) BMP, and 6) varying
amounts of an unlabeled variant BMP-7 molecule, which acts as a
competitor. It is also possible to bind the type I BMP receptor to
the acceptor beads and the type II BMP receptor to the donor
beads.
[0153] In another embodiment, fluorescence assays are used. Either
BMP-7 or a BMP-7 receptor or inhibitor may be labeled with a
fluorescent dye (for examples of suitable dyes, see the Molecular
Probes catalog). As is known in the art, the fluorescence intensity
or anisotropy of a labeled molecule may change upon binding to
another molecule. Fluorescence assays may be performed using 1)
fluorescently labeled BMP-7, 2) a BMP receptor or inhibitor, and 3)
varying amounts of an unlabeled variant BMP-7 protein, which acts
as a competitor.
[0154] In an additional embodiment, scintillation proximity assays
(SPA) are used to determine receptor binding affinity. For example,
BMP receptor-Fc fusions may be bound to protein A coated SPA beads
or flash-plate and treated with S35-labeled BMP; the binding event
results in production of light.
[0155] Cell-Based Activity Assays
[0156] BMPs promote the growth and differentiation of a number of
types of cells. BMP activity may be monitored, for example, by
measuring BMP-induced differentiation of MC3T3-E1 (an
osteoblast-like cell derived from murine calvaria), C3H10T1/2 (a
mouse mesenchymal stem cell line derived from embryonic connective
tissue), ATDC5 (a mouse embryonal carcinoma cell), L-6 (a rat
myoblast cell line) or C2C12 (a mouse myoblastic cell line) cells.
Differentiation may be monitored using, for example, luminescence
reporters for alkaline phosphatase or colorimetric reagents such as
Alcian Blue or PNPP (Asahina et. al. (1996) Exp. Cell Res. 222:
38-47; Inada et. al. (1996) Biochem. Biophys. Res. Commun. 222:
317-322; Jortikka et. al. (1998) Life Sci. 62: 2359-2368; Cheng et.
al. (2003) J. Bone Joint Surgery 95A: 1544-1552). The rat limb bud
cartilage differentiation assay may also be used to monitor
activity in primary cells. In an alternate embodiment, reporter
gene or kinase assays may be used. BMPs activate the JAK-STAT
signal transduction pathway. Accordingly, a BMP responsive cell
line containing a STAT-responsive reporter such as GFP or
luciferase may be used (Kusanagi et. al. (2000) Mol. Biol. Cell.
11: 555-565). In a preferred embodiment, BMP activity in kidney
cells is determined using cell-based assays; see for example Wang
and Hirschberg (2004) J. Biol. Chem. 279: 23200-23206.
[0157] Animal Models of BMP Activity
[0158] In the simplest embodiment, BMP activity in an animal is
measured as bone induction following subcutaneous injection. In a
preferred embodiment, the activity of one or more BMP variants is
determined in an animal model of a BMP-responsive disease or
condition. Animal models of renal disease include, but are not
limited to, the rat nephrotoxic serum nephritis model (Zeisberg et.
al. 2003)), the rat chronic cyclosporine A-induced nephropathy
model (Li et. al. (2004) Am. J. Physiol. Renal Physiol. 286:
F46-57), the mouse unilateral uretreral obstruction model
(Schanstra et. al. (2003) Thromb. Haemost. 89: 735-740),
streptozotocin-induced diabetic nephropathy (Taneda et. al. (2003)
J. Am. Soc. Nephrol. 14: 968-980), the anti-thy 1.1 mAb and Habu
snake venom induced glomerulonephritis models (Dimmler et. al.
(2003) Diagn. Mol. Pathol. 12: 108-117), and the rat 5/6 remnant
kidney model (Romero et. al. (1999) Kidney Int. 55: 945-955).
Animal models of liver disease include, but are not limited to, rat
bile duct ligation/scission model (Park et. al. (2000) Pharmacol.
Toxicol. 87: 261-268), CCl.sub.4 plus ethanol-induced liver damage
(Hall et. al. (1991) Hepatology 12: 815-819),
dimethylnitrosamine-induced liver cirrhosis (Kondou et. al. (2003)
J. Hepatol. 39: 742-748), and thioacetamide-induced liver damage
(Muller et. al. (1988) Exp. Pathol. 34: 229-236). Animal models of
lung disease include, but are not limited to, ovalbumin-induced
airway fibrosis (Kenyon et. al. (2003) Toxicol. Appl. Pharmacol.
186: 90-100), bleomycin-induced lung fibrosis (Izbicki et. al.
(2002) Int. J. Exp. Pathol. 83: 111-119), monocrotaline-induced
pulmonary fibrosis (Hayashi et. al. (1995) Toxicol. Pathol. 23:
63-71), and selective irradiation (Pauluhn et. al. (2001)
Toxicology 161: 153-163). Animal models of neurological disease
include, but are not limited to, animal models for Parkinson's
disease such as the 6-hydroxydopamine (6-OHDA) hemilesioned rat
model and MPTP-induced Parkinson's disease, animal models of ALS
such as rats or mice expressing mutant SOD1 (Shibata et. al. (2002)
Neuropathology 22: 337-349), and animal models of stroke induced by
intracortical microinjection of endothelin or quinolinic acid
(Gilmour et. al. (2004) Behav. Brain Res. 150: 171-183) or cerebral
artery occlusion (Merchenthaler et. al. (2003) Ann. NY Acad. Sci.
1007: 89-100).
[0159] Administration and Treatment Using BMP Variants
[0160] Once made, the BMP variants of the invention may be
administered to a patient to treat a BMP related disorder. The BMP
variants may be administered in a variety of ways, including, but
not limited to orally, parenterally, subcutaneously, intravenously,
intranasally, transdermally, intraperitoneally, intramuscularly,
intrapulmonarally, vaginally, rectally, intranasally or
intraocularly. In some instances, the variant BMP may be directly
applied as a solution or spray.
[0161] The pharmaceutical compositions of the present invention
comprise a BMP variant in a form suitable for administration to a
patient. In the preferred embodiment, the pharmaceutical
compositions are in a sterile, water-soluble form and may include
pharmaceutically acceptable acid addition salts or pharmaceutically
acceptable base addition salts. The pharmaceutical compositions may
also include one or more of the following: carrier proteins such as
serum albumin; buffers such as NaOAc; fillers such as
microcrystalline cellulose, lactose, corn and other starches;
binding agents; sweeteners and other flavoring agents; coloring
agents; and polyethylene glycol. Additives that are "generally
recognized as safe" (GRAS) are well known in the art, and are used
in a variety of formulations.
[0162] Any of a number of drug delivery devices or sustained
release formulations may be used. For example, a variant BMP may be
administered as a pro-protein comprising a BMP pro-domain and a BMP
mature domain. BMPs may also be administered as BMP-impregnated
matrix material (for example Geiger et. al. Adv. Drug Deliv. Rev.
(2003) 55: 1613-1629; Hu et. al. J. Biomed. Mater. Res. (2003) 67A:
591-598; Peel et. al. J. Craniofac Surg. (2003) 14: 284-291); such
a method of administration is especially preferred for promoting
bone healing and growth. Furthermore, implants for bone repair may
be coated with BMPs to promote bone healing and improve bone
strength (Schmidmaier et. al. Bone (2002) 30: 816-822). In a
further embodiment, the variant BMPs are added in a micellular
formulation (U.S. Pat. No. 5,833,948), liposomes (Matsuo et. al. J.
Biomed. Mater. Res. (2003) 66A: 747-754), biodegradable polymers
(Saito and Takaoka, Biomaterials (2003) 24: 2287-2293; Saito et.
al. Bone (2003) 32: 381-386; Weber et. al. Int. J. Oral Maxillofac.
Surg. (2002) 31: 60-65; Saito et. al. J. Bone Joint Surg. Am.
(2001) 83-A: S92-S98), hydrogels (Yamamoto et. al. Biomaterials
(2003) 24: 4375-4383), or the like.
[0163] Combinations of pharmaceutical compositions may be
administered. Moreover, the compositions may be administered in
combination with other therapeutics.
[0164] Nucleic acid encoding the variant BMPs may also be used in
gene therapy. In gene therapy applications, genes are introduced
into cells in order to achieve in vivo synthesis of a
therapeutically effective genetic product, for example for
replacement of a defective gene. "Gene therapy" includes both
conventional gene therapy where a lasting effect is achieved by a
single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA. Any of a variety of
techniques known in the art may be used to introduce nucleic acids
to the relevant cells. The oligonucleotides may be modified to
enhance their uptake, e.g. by substituting their negatively charged
phosphodiester groups by uncharged groups. For review of gene
marking and gene therapy protocols see Anderson et al., Science
256:808-813 (1992).
EXAMPLES
Example 1
Structural Modeling
[0165] Hexameric complexes comprising a BMP-7 dimer or a BMP-2
dimer bound to two ALK-3 receptors and two ActRIIa receptors was
constructed using the structure of BMP-7 bound to ActRIIa (PDB code
1LX5) and the structure of BMP-2 bound to ALK-3 (PDB code 1ES7).
Using InsightII (Accelrys), the BMP structures were superimposed as
follows: BMP-2 residues 22-32 superimposed with BMP-7 residues
47-56, BMP-2 residues 49-71 superimposed with BMP-7 residues 73-95,
and BMP-2 residues 101-106 superimposed with BMP-7 residues
126-131. This yielded a backbone atom RMSD of 0.77 .ANG.. The
superposition was repeated so that chain A in the BMP-7 structure
was superimposed onto chains A and C of the BMP-2 structure. The
sequence alignment between BMP-2 and BMP-7 is shown in FIG. 3 and
the structure of the hexameric complex is shown in FIG. 2.
[0166] Homology modeling was used to generate structures of
additional BMP receptors bound to BMP-2 and BMP-7. As shown in FIG.
5, the sequences of ALK-2 and ALK-6 were aligned with ALK-3, and
the sequences of ActRIIb and BMPRII were aligned with ActRIIa. The
Modeler tool in InsightII (Accelrys) was used to generate the
homology models. Disulfide pairs were manually constrained as
follows (using the crystallographic numbering from 1LX5 and 1ES7):
Alk3: 238-259, 240-244, 253-277, 287-301, 302-307; ActRIIa: 11-41,
31-59, 66-85, 72-84, and 86-91. Three models were generated for
each molecule; the model with the best energy and --InPDF score was
selected for subsequent PDA.RTM. technology calculations. Homology
modeling was also used to generate structures of BMP-4, BMP-5,
BMP-6, and BMP-8. BMP-4 was modeled using the BMP-2 structure while
BMP-5, BMP-6, and BMP-8 were modeled using the BMP-7 structure. The
BMP sequences were aligned as shown in FIG. 3 (SEQ ID NOS:7-12).
PDA.RTM. technology calculations were used to model the BMP-4,
BMP-5, BMP-6, and BMP-8 structures.
Example 2
Identification of Exposed Hydrophobic Residues in BMPs
[0167] Structures of BMP-7 dimer ("dimer") and BMP-7 dimer bound to
ALK-3 and ActRIIa "hexamer") were analyzed to identify
solvent-exposed hydrophobic residues. The absolute and fractional
solvent-exposed hydrophobic surface area of each residue was
calculated using the method of Lee and Richards (J. Mol. Biol. 55:
379-400 (1971)) using an add-on radius of 1.4 .ANG. (Angstroms).
Each residue was also classified as core, boundary, or surface (see
Dahiyat and Mayo Science 278: 82-87 (1997)).
[0168] Solvent exposed hydrophobic residues in BMP-7 were defined
to be hydrophobic residues with at least 50 .ANG..sup.2 (square
Angstroms) exposed hydrophobic surface area in the BMP-7 dimer (PDB
code 1LX5, chain A, plus symmetry-related BMP-7 molecule). Exposed
hydrophobic surface area was also measured in the context of the
BMP-7/ALK-2/ActRIIa hexamer and RESCLASS was run to categorize each
position as core, boundary, or surface. TABLE-US-00001 TABLE 1
Exposed hydrophobic residues in BMP-7. hexamer dimer hexamer # wt
dimer RC RC expH expH 44 TYR surface surface 85.5 95.5 52 TRP
boundary core 82.9 5.9 55 TRP boundary boundary 168.4 125.5 57 ILE
boundary core 70.3 29.8 73 PHE surface core 70.5 64.6 78 TYR
surface core 107.9 71.2 86 ILE surface core 60.7 4.3 90 LEU
boundary core 51.7 10.6 93 PHE boundary core 113.1 10.9 94 ILE
boundary core 79.0 37.3 115 LEU surface core 56.9 0.0 116 TYR
boundary core 51.5 51.6 117 PHE surface boundary 97.5 25.4 123 VAL
surface core 86.0 14.4 125 LEU surface core 88.4 60.9 128 TYR
boundary core 64.9 17.4
Example 3
Identification of Receptor and Inhibitor Interface Residues in
BMP-7
[0169] Potential sites of interactions between BMP-7 and ALK-3,
BMP-7 and ActRIIa, and BMP-7 and noggin were identified by
examining the structure of the hexameric structure described in
Example 1 and the co-crystal structure of BMP-7 and noggin (PDB
code 1M4U). Next, distance measurements were used to identify
residues that may participate in intermolecular interactions.
Residues in BMP-7 that are within 5 .ANG. (Angstroms) of the ALK-3,
ActRIIa, or noggin interfaces (as measured by CA-CA distances) are
shown below, along with the receptor or inhibitor positions that
are contacted. Next, the receptor sequence alignments used for
homology modeling were analyzed for polymorphisms. Information
about receptor polymorphisms was used to design receptor-specific
variants, described below. If the receptor positions are
polymorphic, it is noted in Table 2; "na" indicates that the
receptor positions were not sufficiently well-aligned to
unambiguously identify the polymorphisms. However,
receptor-specific BMP variants may be identified that contact such
unaligned regions of the receptors. TABLE-US-00002 TABLE 2 BMP-7
receptor and inhibitor contacts Contacts: A, B = ALK-3; # Wt D, F =
ActRIIa; and N = noggin receptor polymorphisms 39 LYS ASP A 246 246
(ALK6 = E, ALK3 = D, ALK2 na) 44 TYR ASN D 65, ILE D 64,
65(ActRIIa, ActRIIb N, BMPRII na), ASP D 63 64(ActRIIb = F, ActRIIa
= I, BMPRII na), 63(ActRIIa, ActRIIb D, BMPRII na) 47 PHE PHE B 285
285(ALK6, ALK3 = F, ALK2 = M) 48 ARG LYS D 76, GLN N 208,
76(ActRIIb = E, ActRIIa = K, BMPRII = T) ARG N 209, ARG N 210 49
ASP LYS B 292 na 50 LEU SER B 290, PHE B 285 290(ALK6 = T, ALK3 =
S, ALK2 = P), 285(ALK6, ALK3 = F, ALK2 = M) 51 GLY PRO B 291, SER B
290, 291 P conserved, 290(ALK6 = T, LYS B 292 ALK3 = S, ALK2 = P),
292 na 52 TRP PHE B 285, LYS B 288, 285(ALK6, ALK3 = F, ALK2 = M),
SER B 290, PRO B 291, 288(ALK6 = R, ALK3, ALK2 = K), ILE N 33, ARG
N 34, 290(ALK6 = T, ALK3 = S, ALK2 = P), 291 PRO N 35 P conserved
53 GLN LYS D 76, ARG N 206 76 (ActRIIb = E, ActRIIa = K, BMPRII =
T) 54 ASP LYS B 288, GLU D 80, 288(ALK6 = R, ALK3, ALK2 = K), ARG N
206, GLN N 208 80(ActRIIb = Q, ActRIIa = E, BMPRII na) 55 TRP LYS B
288, ARG N 34, 288(ALK6 = R, ALK3, ALK2 = K) PRO N 35 56 ILE PHE B
285 285(ALK6, ALK3 = F, ALK2 = M) 57 ILE PHE D 83, VAL D 81, 83 F
conserved, 81(ActRIIa, ActRIIb THR D 44, ARG N 204, V, BMPRII na),
44(ActRIIb = S, ARG N 206, ILE N 218 ActRIIa = T, BMPRII = L) 58
ALA PHE D 83, TRP D 60, 60 W conserved, 83 F conserved LEU N 46,
GLU N 48, ARG N 204 59 PRO ASP D 63, ASN D 65, 63(ActRIIa, ActRIIb
D, BMPRII na), TRP D 60, PHE D 83, 65(ActRIIa, ActRIIb N, BMPRII
na), LEU N 46, ILE N 47 60 W conserved, 83 F conserved 60 GLU LYS D
76, ASN D 65, 76(ActRIIb = E, ActRIIa = K, BMPRII = T), GLU D 74,
PHE N 54 65(ActRIIa, ActRIIb N, BMPRII na), 74(ActRIIb = A, ActRIIa
= E, BMPRII = V) 61 GLY ASN D 65 65(ActRIIa, ActRIIb N, BMPRII na)
62 TYR ASP D 63, ASN D 65, 63(ActRIIa, ActRIIb D, BMPRII na), ILE D
64 65(ActRIIa, ActRIIb N, BMPRII na), 64(ActRIIb = F, ActRIIa = I,
BMPRII na) 63 ALA ILE D 64 64(ActRIIb = F, ActRIIa = I, BMPRII na)
73 PHE ARG A 297, GLU A 264, 297(ALK6, ALK3 = R, ALK2 = Q) ILE N 33
264(ALK6, ALK3 = E, ALK2 = S) 74 PRO HIS A 243, ILE A 262,
243(ALK3, ALK6 H, ALK2 na), ILE A 299, PHE A 260, 262(ALK6 = M,
ALK3 = I, ALK2 = S), GLU A 264, GLN A 286, 299(ALK6, ALK3 = I ALK2
= V), 260 F MET A 278, LEU N 31, conserved, 264(ALK6, ALK3 = E, ILE
N 33 ALK2 = S), 286(ALK6, ALK3 = Q ALK2 = T), 278(ALK6 = L, ALK3 =
M, ALK2 = F) 75 LEU GLN A 286, TYR N 30, 286(ALK6, ALK3 = Q ALK2 =
T) LEU N 31, HIS N 32, ILE N 33 76 ASN HIS A 243, PHE A 260,
243(ALK3, ALK6 H, ALK2 na), 260 F GLY A 276, MET A 278, conserved,
276 G conserved, PRO A 245, CYS A 277, 278(ALK6 = L, ALK3 = M, ALK2
= F), TYR N 30, LEU N 31, 245(ALK3, ALK6 P, ALK2 na), 277 C HIS N
32 conserved 77 SER CYS A 277, CYS A 253, 277 C conserved, 253 C
conserved, THR A 255, PHE A 260, 255(ALK3, ALK6 T, ALK2 na), 260 F
LYS A 279, GLY A 276, conserved, 279(ALK6 = G, ALK3 = K, MET A 278,
PRO A 245, ALK2 na), 276 G conserved, MET N 27, HIS N 29, 278(ALK6
= L, ALK3 = M, ALK2 = F), TYR N 30, HIS N 32 245(ALK3, ALK6 P, ALK2
na) 78 TYR ASP A 246, PRO A 245, 246(ALK6 = E, ALK3 = D, ALK2 na),
ASP A 247 245(ALK3, ALK6 P, ALK2 na), 247(ALK3, ALK6 D, ALK2 na) 80
ASN LYS A 279 279(ALK6 = G, ALK3 = K, ALK2 na) 83 ASN GLU A 281,
GLY A 282, 281(ALK3, ALK6, 282 ALK2 na), PHE A 285, ARG N 34,
282(ALK3, ALK6 G, ALK2 na), PRO N 35, ALA N 36 285(ALK6, ALK3 = F
ALK2 = M) 86 ILE GLN A 286, GLY A 282, 286(ALK6, ALK3 = Q ALK2 =
T), PHE A 285, HIS N 32, 282(ALK3, ALK6 G, ALK2 na), ILE N 33, ARG
N 34, 285(ALK6, ALK3 = F ALK2 = M) PRO N 35 87 VAL PRO N 35 90 LEU
ASP A 289, SER A 290, 289(ALK6, ALK3 = D ALK2 = T), PHE A 285, ARG
A 297, 290(ALK6 = T ALK3 = S ALK2 = P), GLN A 286, ILE N 33
285(ALK6, ALK3 = F ALK2 = M), 297(ALK6, ALK3 = R ALK2 = Q),
296(ALK3, ALK6 R, ALK2 na) 93 PHE ALA A 293, ARG A 297, 293(na),
295(na), 294(na), ASP A 289, LEU A 295, 297(ALK6, ALK3 = R ALK2 =
Q), SER A 290, GLN A 294, 289(ALK6, ALK3 = D ALK2 = T), GLU A 264
290(ALK6 = T ALK3 = S ALK2 = P), 264(ALK6, ALK3 = E ALK2 = S) 94
ILE SER A 290, LYS A 292, 290(ALK6 = T ALK3 = S ALK2 = P), ALA A
293 292(na), 293(na) 108 GLN ASP D 36 36(ActRII, ActRIIb D, BMPRII
N) 110 ASN LYS D 37, ASP D 62, 36(ActRII, ActRIIb D, BMPRII ASP D
36 N), 37(ActRII, ActRIIa K, BMPRII na), 62 D conserved 111 ALA LEU
D 61, LYS D 37 61(ActRIIa, ActRIIb L, BMPRII G), 37(ActRII, ActRIIa
K, BMPRII na) 112 ILE LEU D 61 61(ActRIIa, ActRIIb L, BMPRII G) 113
SER LEU D 61, TRP D 60, 61(ActRIIa, ActRIIb L, BMPRII G), 60 LEU N
43, VAL N 44, W conserved ASP N 45, LEU N 46 114 VAL TRP D 60, LEU
N 46 60 W conserved 115 LEU PHE D 83, TRP D 60, 83 F conserved, 60
W conserved, 56 PHE D 42, THR D 44, K conserved, 42(ActRIIb, BMPRII
= Y, LYS D 56, LEU N 46, ActRIIa = F), 44(ActRIIb = S, ActRIIa = T,
PHE N 168, ARG N 204, BMPRII = L) ILE N 220 116 TYR ASP B 284, PRO
N 37 284(ALK6, ALK3 = D ALK2 = K) 117 PHE VAL D 81, GLU D 80,
81(ActRIIa, ActRIIb V, BMPRII na), ARG N 206, ILE N 218 80(ActRIIb
= Q, ActRIIa = E, BMPRII na) 119 ASP HIS N 29 122 ASN ARG D 20, ASN
D 17, 20(ActRIIb = L, ActRIIa = K, BMPRII GLN N 221 na),
17(ActRIIa, ActRIIb N, BMPRII na) 123 VAL VAL D 81, VAL D 55,
81(ActRIIa, ActRIIb V, BMPRII na), LYS D 56, THR D 44, 55 V
conserved, 56 K conserved, LYS D 46, ILE N 218, 44(ActRIIb = S,
ActRIIa = T, BMPRII = L), PRO N 219, ILE N 220, 46(ActRIIb = A,
ActRIIa = K, GLN N 221 BMPRII = E), 124 ILE HIS N 199, GLN N 221
125 LEU TRP D 60, PHE D 42, 60 W conserved, 56 K conserved, LYS D
56, LEU N 43, 42(ActRIIb, BMPRII = Y, ActRIIa = F) ASP N 45, LEU N
46, GLN N 221, TYR N 222, PRO N 223 126 LYS TYR B 280, GLU B 281,
280(ALK6 = L ALK3 = Y ALK2 na), ASP B 284, PRO N 37, 281(ALK2 na),
284(ALK6, ALK3 = D SER N 38, ASP N 39, ALK2 = K) LEU N 43, HIS N
199 127 LYS LEU D 61, LYS D 37, 37(ActRII, ActRIIa K, BMPRII na),
ALA N 36, PRO N 37, 61(ActRIIa, ActRIIb L, BMPRII G), SER N 38, ASN
N 40, 36(ActRII, ActRIIb D, BMPRII N) LEU N 41, PRO N 42, LEU N 43
128 TYR ASP B 284, PHE B 285, 284(ALK6, ALK3 = D ALK2 = K), PRO N
35, ALA N 36, 285(ALK6, ALK3 = F ALK2 = M) PRO N 37, SER N 38 129
ARG GLU B 281, ASN E 83, 281(ALK3, ALK6 E, ALK2 na) ALA N 36, SER N
38, ASN N 40 130 ASN GLU B 281 281(ALK3, ALK6 E, ALK2 na) 131 MET
PHE B 285, PRO N 35 285(ALK6, ALK3 = F ALK2 = M) 134 ARG ASP D 36
36(ActRII, ActRIIb D, BMPRII N)
Example 4
Identification of Regions of High Electrostatic Potential in
BMP-7
[0170] The electrostatic potential at each position in BMP-7 was
determined using the Dybye-Huckel equation in the context of the
BMP-7 dimer. Positions with electrostatic potential greater than
0.5 or less than -0.5 are listed in the table below; modifications
at these positions may confer increased stability or receptor
binding specificity. TABLE-US-00003 TABLE 3 Regions of high
electrostatic potential in BMP-7 Residue Residue Electrostatic
number name potential 46 SER -0.72 67 CYS 0.73 68 GLU 0.50 69 GLY
0.58 70 GLU 0.55 71 CYS 0.50 72 ALA 0.62 82 THR 0.57 105 ALA 0.54
106 PRO 0.67 107 THR 0.65 108 GLN 0.68 109 LEU 0.79 110 ASN 1.00
111 ALA 1.51 113 SER 0.68 122 ASN -0.64 133 VAL 0.68 135 ALA 0.53
136 CYS 0.62
Example 5
Identification of Preferred Substitutions to BMPs
[0171] Analogous contact environment (ACE) calculations, were
performed on BMP-7 using complete PFAM alignment for BMP-7. ACE
calculations identify alternate residues for each position that are
observed in structurally similar contexts in homologous proteins.
The calculations were performed using a low stringency threshold of
0.8 and a high stringency threshold of 0.5. TABLE-US-00004 TABLE 4
Residues observed in analogous structural contexts in BMP-7
homologs. ACE, low ACE, high residue Wt stringency stringency 36
GLN E Q T Q T 37 ALA A G S V A G 38 CYS C C 39 LYS K R K 40 LYS K R
T K 41 HIS H K R H 42 GLU E S E 43 LEU F L M P L P 44 TYR F Y F Y
45 VAL I R V V 46 SER D E N S S 47 PHE E L S F 48 ARG K Q R K Q R
49 ASP A D E Q D 50 LEU F I L M V L V 51 GLY D G N G N 52 TRP W W
53 GLN D H L N Q R S D H L N Q S 54 ASP D N R D N 55 TRP W W 56 ILE
I V I 57 ILE I V I 58 ALA A K Q S Y A 59 PRO P P 60 GLU A E G H K M
A E K M P M P Q R S T Q R S 61 GLY G G 62 TYR F Y Y 63 ALA A D E G
H A M Q S M N Q S 64 ALA A G A 65 TYR F N Y F N Y 66 TYR F Y Y 67
CYS C C 68 GLU A D E H K Q R D E 69 GLY G G 70 GLU E E 71 CYS C C
72 ALA A D N P S V A N S V 73 PHE F F 74 PRO P P 75 LEU L L 76 ASN
A D N S N 77 SER A S A S 78 TYR C F H Y C F H Y 79 MET A M M 80 ASN
N N 81 ALA A F G P S T A 82 THR T T 83 ASN K N S N 84 HIS H H 85
ALA A A 86 ILE I L V I 87 VAL I L M V V 88 GLN K Q Q 89 THR L T T
90 LEU L L 91 VAL V V 92 HIS H N H 93 PHE A F L S F S 94 ILE F I I
95 ASN N N 96 PRO P P 97 GLU A D E G K N D E Q R S 98 THR A K R T T
Y 99 VAL T V V 100 PRO G P P 101 LYS K L Q K Q 102 PRO A P S T V W
P 103 CYS C K C 104 CYS C W C 105 ALA A G H I Q R A S T V 106 PRO N
P P 107 THR T T 108 GLN K Q K Q 109 LEU L L 110 ASN H N N 111 ALA A
G S A 112 ILE I L T I 113 SER P S T P S 114 VAL I L M V L M V 115
LEU L L 116 TYR F Y Y 117 PHE F I K L Q Y F Y 118 ASP D D 119 ASP D
E N S D E N S 120 SER D E G H N S N S 121 SER A D E H K N R S A D S
122 ASN A N S N 123 VAL I L V V 124 ILE I V I V 125 LEU I K L Y L Y
126 LYS K N R Y K R
[0172] PDA.RTM. technology calculations were performed to identify
alternate residues that are compatible with the structure and
function of BMP-7. At each variable position, energies were
calculated for the wild type residue and alternate residues with
decreased hydrophobic or increased polar character. First, point
mutation calculations were run for each template. The energy of
each alternate amino acid in its most favorable rotameric
conformation was compared to the energy of the wild type residue in
the crystallographically observed rotameric conformation; all
reported energies in the table below are [E(wild type)-E(variant)].
For residues that are within 5 .ANG. of at least one atom in the
type I or type II receptor, calculations were also performed using
templates consisting of the BMP-7 dimer bound to receptor.
TABLE-US-00005 TABLE 5 Energies of most favorable alternate
residues in each variable position in BMP-7 # Wt dimer ALK2 ALK3
ALK6 ActRIIa ActRIIb 36 GLN N: -4.8 N: 0.4 N: -6.6 D: -3.7 Q: 0.8
Q: -5.6 S: -3.0 D: 0.8 D: -5.2 39 LYS E: -11.9 T: 0.0 T: 1.4 S: 2.7
E: -2.0 E: -1.8 K: -10.5 E: 0.2 A: 1.6 E: 3.3 S: -0.4 K: -0.3 Q:
-10.0 S: 0.8 S: 2.6 T: 4.1 K: -0.2 Q: 0.4 42 GLU Q: -3.2 Q: -5.0 Q:
-6.2 E: -2.3 E: -4.0 R: -5.7 N: -0.9 N: -2.9 E: -5.5 44 TYR Q: -6.6
Q: -3.5 Q: -3.5 Q: -3.5 Q: 0.8 Q: -3.1 E: -5.6 E: -2.6 E: -2.6 E:
-2.6 R: 1.1 R: -2.5 N: -4.6 N: -1.5 D: -1.5 N: -1.5 E: 1.9 E: -2.2
48 ARG E: -5.4 N: -3.3 Q: -3.9 N: -3.2 N: -8.4 N: -3.9 Q: -4.3 Q:
-3.2 N: -3.3 Q: -2.9 Q: -7.5 Q: -3.7 N: -3.8 R: -2.6 R: -2.9 R:
-2.5 D: -7.1 E: -2.9 49 ASP S: -1.4 D: -0.3 N: -3.4 D: -0.6 R: 0.0
D: -2.5 N: -0.5 Q: 0.2 Q: -1.9 52 TRP Q: 2.3 K: 14.5 E: 14.5 E:
14.5 K: 1.8 K: 0.6 K: 3.2 E: 15.0 K: 15.0 K: 15.0 Q: 2.3 Q: 1.4 E:
3.4 A: 16.7 Q: 16.7 Q: 16.7 E: 3.5 E: 2.6 53 GLN D: -6.1 D: 6.2 A:
7.0 D: 5.9 E: -7.5 D: -6.9 A: -5.7 A: 6.3 H: 7.3 A: 6.4 S: -6.3 S:
-6.4 S: -5.7 T: 6.6 T: 7.8 H: 6.6 Q: -6.2 Q: -6.4 54 ASP D: -0.3 N:
-6.4 N: -6.4 N: -6.3 N: -3.6 N: 0.0 S: 0.2 D: -5.5 D: -5.6 D: -5.5
Q: -3.4 D: 0.7 N: 0.6 Q: -4.9 Q: -4.5 Q: -5.4 D: -2.8 Q: 1.4 55 TRP
N: -19.6 R: 11.3 H: 11.3 H: 11.3 Q: -18.3 Q: -17.4 R: -19.3 H: 13.0
S: 13.0 Q: 13.0 N: -16.7 E: -15.9 D: -18.8 Q: 13.9 T: 13.9 R: 13.9
E: -16.6 N: -15.9 57 ILE E: 1.0 T: -4.9 T: -4.9 T: -4.9 E: 9.0 E:
8.8 K: 1.1 D: -3.1 D: -3.1 D: -3.1 T: 11.6 D: 9.3 Q: 2.2 R: -2.5 Q:
-2.5 R: -2.5 D: 12.0 T: 9.4 60 GLU Q: -1.9 E: -1.8 E: 0.6 E: -1.7
E: 0.2 T: -0.3 N: -1.4 Q: 3.9 Q: 4.5 Q: 4.1 T: 1.7 E: 0.2 R: -1.3
N: 4.5 N: 5.3 N: 4.7 D: 2.8 K: 0.8 63 ALA R: -5.1 A: 0.0 E: -4.0 Q:
-4.8 S: 0.8 Q: -1.6 E: -3.9 T: 2.2 A: 0.2 65 TYR A: 7.9 E: 8.5 H:
8.5 70 GLU E: 2.8 D: -2.3 E: -5.6 Q: 4.7 Q: -1.5 Q: -3.0 D: 4.9 E:
-0.7 T: 0.4 73 PHE R: 0.4 H: 11.2 H: 11.2 H: 11.2 H: -6.5 H: -6.5
Q: 1.8 E: 18.6 A: 18.6 D: 18.6 D: -5.7 D: -5.7 E: 2.4 D: 21.2 S:
21.1 A: 21.1 S: -4.0 A: -4.1 76 ASN N: -6.6 A: -3.9 A: -5.2 N: -4.0
D: -6.2 S: -4.5 Q: -6.4 T: -2.2 S: -2.4 S: -1.2 Q: -5.3 Q: -4.4 D:
-6.4 S: -1.1 T: 3.5 T: 1.6 N-5.2 D: -4.2 77 SER N: -5.7 S: -1.8 A:
-4.5 K: -3.0 N: -8.9 N: -6.9 D: -4.6 A: 1.3 S: -0.2 D: -0.9 D: -7.9
D: -5.8 S: -3.9 T: 10.1 T: 0.1 A: -0.3 S: -6.8 S: -4.8 78 TYR N:
-15.6 S: 3.2 S: 3.2 S: 3.2 N: -13.2 R: -12.8 D: -14.6 D: 6.4 A: 6.4
T: 6.4 D: -12.8 N: -12.4 S: -13.9 Q: 6.5 H: 6.5 A: 6.5 S: -11.6 D:
-12.1 86 ILE K: -5.0 T: 9.9 T: 9.9 D: 9.9 E: -3.6 K: -5.1 E: -4.3
D: 10.9 D: 10.9 A: 10.9 K: -2.2 E: -3.6 Q: -4.0 A: 11.6 A: 11.6 T:
11.6 Q: -1.9 Q: -2.1 88 GLN E: -0.1 T: 1.6 Q: 2.4 90 LEU K: -3.3 E:
6.3 E: 6.3 E: 6.3 E: -0.8 E: -2.2 E: -0.6 D: 9.4 T: 9.4 T: 9.4 Q:
-0.7 Q: -2.1 Q: 0.5 T: 9.5 D: 9.5 D: 9.5 R: -0.6 R: -0.1 93 PHE S:
-18.0 E: 0.3 S: 0.3 E: 0.3 D: -15.3 D: -15.1 D: -17.4 T: 2.3 D: 2.3
D: 2.3 S: -15.1 S: -14.8 R: -17.4 D: 3.0 E: 3.0 Q: 3.0 N: -13.8 N:
-13.6 94 ILE K: -2.7 E: 4.1 T: 4.1 H: 4.1 E: -2.0 K: -2.0 E: -2.0
D: 4.3 N: 4.3 D: 4.7 Q: -.06 E: -1.9 Q: -0.8 A: 4.7 D: 4.7 E: 5.9
K: -0.4 Q: -0.4 95 ASN Q: 3.4 N: 5.9 Q: -2.6 D: 3.9 D: 6.6 N: 0.3
S: 4.4 Q: 7.3 D: 0.4 97 GLU N: -5.8 -- N: -3.6 D: -4.9 D: -2.6 S:
-4.1 S: -2.6 98 THR D: -0.8 N: -2.1 Q: -9.3 E: -0.7 D: -1.4 R: -8.3
K: -0.3 Q: -1.1 E: -8.0 108 GLN N: -5.7 Q: -2.6 S: -6.8 D: -5.2 D:
-0.7 N: -5.8 Q: -5.1 S: 0.4 D: -4.8 110 ASN E: 1.0 A: -7.1 Q: -8.1
Q: 4.0 E: -5.7 E: -6.7 S: 8.3 D: -4.6 A: -4.6 111 ALA A: 0.0 A:
-4.7 A: -6.4 S: 1.7 S: -2.1 S: -3.3 H: 9.2 T: 26.2 D: 46.3 115 LEU
K: -3.4 K: -3.8 E: -3.8 K: -3.8 E: 8.9 E: 6.7 E: -3.2 E: -3.3 K:
-3.3 E: -3.3 T: 11.5 A: 10.1 D: -1.3 D: -1.5 D: -1.5 D: -1.5 D:
12.1 Q: 10.4 116 TYR H: 4.5 H: 4.6 H: 4.6 H: 4.6 H: 1.7 H: 1.5 S:
6.0 T: 8.8 T: 8.8 A: 8.8 T: 4.3 T: 4.6 T: 7.5 A: 9.3 A: 9.3 S: 9.3
S: 6.4 S: 6.7 117 PHE Q: -7.3 K: -4.1 R: -4.1 R: -4.1 K: 6.7 H: 7.1
R: -7.2 Q: -3.9 K: -3.9 Q: -3.9 E: 9.1 K: 8.0 E: -6.9 R: -3.7 Q:
-3.7 K: -3.7 H: 10.3 E: 8.1 119 ASP R: -2.5 N: -0.4 N: -5.2 Q: -2.3
S: 0.7 S: -4.1 N: -1.9 D: 0.8 D: -3.8 120 SER N: -5.8 S: -1.7 N:
-7.4 S: -4.6 N: -1.6 Q: -6.6 D: -4.4 D: -0.1 S: -5.9 121 SER N:
-4.7 N: -4.8 Q: -7.0 Q: -4.0 D: -3.4 E: -6.2 S: -3.7 Q: -3.2 K:
-5.9 122 ASN R: -2.6 N: 5.3 Q: -6.0 R: 0.0 N: -2.5 Q: 6.3 E: -5.7
Q: 0.6 Q: -2.4 D: 7.6 R: -5.6 N: 0.7 123 VAL Q: -5.4 T: -9.2 T:
-9.2 T: -9.2 D: 4.8 T: 4.8 E: -4.5 R: -8.6 R: -8.6 R: -8.6 T: 5.1
A: 6.0 S: -4.1 E: -8.3 E: -8.3 E: -8.3 A: 6.4 D: 7.6 125 LEU Q:
-10.7 Q: -9.0 Q: -9.0 Q: -9.0 H: -1.0 H: -0.6 E: -9.8 E: -8.4 E:
-8.4 E: -8.4 A: 3.8 E: 3.2 S: -9.3 S: -7.3 S: -7.3 S: -7.3 D: 3.8
K: 3.2 126 LYS T: -1.2 D: -10.8 Q: -2.2 D: -4.6 Q: -9.9 Q: -9.2 D:
1.0 S: -7.4 R: -1.5 E: -4.6 E: -9.3 E: -8.5 E: 1.0 N: -5.9 D: -1.3
K: -4.2 R: -7.9 R: -7.3 127 LYS Q: -13.7 N: -17.3 Q: -7.9 N: -17.8
D: -12.5 T: -5.2 E: -12.8 D: -16.7 E: -7.2 D: -17.5 T: -11.7 S:
-2.5 R: -12.0 Q: -13.7 S: -5.9 Q: -13.5 S: -10.2 D: -0.9 128 TYR K:
1.2 E: 4.9 H: 4.9 H: 4.9 E: 2.5 E: 0.7 E: 2.3 A: 7.1 D: 9.9 K: 9.9
D: 3.9 D: 3.6 Q: 4.1 H: 9.9 K: 10.5 D: 10.5 K: 5.9 K: 5.2 129 ARG
Q: -21.5 D: -8.3 D: -4.3 D: -6.6 N: -10.3 E: -9.9 E: -21.1 E: -6.6
S: -4.1 S: -6.6 D: -9.7 N: -8.9 N: -19.1 N: -6.0 Q: -2.9 Q: -6.1 E:
-9.4 Q: -8.3 130 ASN E: -0.9 D: -4.3 D: -2.1 D: -2.0 Q: -0.7 E:
-3.5 R: -1.7 R: -1.7 R: -0.4 Q: -3.3 E: -1.7 Q: -0.9 134 ARG Q:
-7.6 Q -6.7 Q: -4.0 S: -1.1 R: -0.8 R: -1.0 E: -7.1 E: -6.0 D: -3.8
D: -1.0 S: 0.0 S: 0.5 R: -6.5 R: -5.2 S: -3.7 Q: -1.0 D: 1.3 D: 1.7
135 ALA E: -3.4 D: -3.4 Q: -3.0
[0173] Next, combinatorial calculations were performed in which
multiple variable positions located close in space were allowed to
vary. The most favorable amino acid sequence was first identified
with DEE, and then Monte Carlo calculations were performed to
identify 10,000 favorable energies. All residues that were selected
for a given position in at least 500 of the top 10,000 sequences
were noted, and the number of occurrences is given in the table
below. For residues that are within 5 .ANG. of at least one atom in
the type I or type II receptor, calculations were also performed
using templates consisting of the BMP-7 dimer bound to receptor.
TABLE-US-00006 TABLE 6 Preferred alternate residues identified
using combinatorial PDA .RTM. calculations # wt dimer ALK2 ALK3
ALK6 ActRII ActRIIb 36 GLN Q: 7809 M: 9996 Q: 10000 N: 1884 39 LYS
S: 9842 S: 8163 S: 9997 D: 5917 S: 10000 S: 10000 D: 1118 M: 3085
T: 572 42 GLU D: 4594 D: 4405 L: 9999 N: 4513 F: 3105 L: 893 N:
2490 48 ARG H: 9950 R: 9994 Q: 7431 R: 10000 E: 9274 R: 6089 E:
2530 Q: 649 Q: 1849 N: 1072 K: 936 49 ASP S: 5123 R: 10000 N: 8591
N: 4554 M: 766 R: 589 52 TRP W: 9937 W: 10000 W: 10000 W: 10000 W:
10000 W: 10000 53 GLN Q: 5056 D: 9292 W: 6190 D: 9673 S: 9247 R:
9596 E: 4894 Q: 695 D: 3771 Q: 753 54 ASP D: 9422 N: 7578 N: 9326
S: 578 D: 1217 S: 606 S: 1205 55 TRP F: 9234 W: 10000 W: 10000 W:
10000 K: 7426 Q: 4838 Q: 2063 K: 2656 N: 1596 57 ILE Q: 4803 V:
7154 V: 9029 T: 6435 I: 10000 I: 10000 E: 4362 T: 1771 E: 940 V:
2518 Q: 928 60 GLU K: 4894 Q: 6658 R: 6190 Q: 8619 E: 10000 E: 9727
Q: 4752 E: 1911 Q: 3243 E: 878 N: 1072 70 GLU E: 10000 M: 7656 E:
10000 E: 1051 T: 840 73 PHE M: 9998 F: 9569 F: 10000 F: 9968 76 ASN
N: 7622 A: 9496 A: 10000 A: 9309 D: 8520 D: 9528 D: 2269 S: 504 S:
658 K: 1480 77 SER N: 9226 A: 9989 D: 10000 D: 10000 N: 4869 N:
5705 S: 2426 D: 2067 D: 2247 S: 1703 86 ILE L: 5322 V: 3799 I:
10000 D: 4757 F: 4678 M: 2956 I: 3625 I: 2197 H: 737 D: 757 V: 548
90 LEU I: 7320 L: 7909 I: 10000 L: 8453 L: 1971 I: 2091 I: 1547 93
PHE R: 10000 M: 9971 F: 10000 F: 9422 94 ILE K: 6521 I: 10000 I:
8908 I: 5328 I: 3472 V: 705 V: 3624 H: 636 95 ASN N: 10000 N: 10000
E: 6756 M: 2423 R: 821 97 GLU E: 8909 R: 6746 N: 565 W: 1317 F: 968
E: 681 98 THR K: 9813 E: 9849 R: 8142 K: 1037 Q: 659 108 GLN W:
10000 W: 5051 E: 8219 L: 4949 M: 1368 115 LEU E: 9073 E: 5297 E:
9835 E: 5464 I: 9494 I: 8877 K: 1647 K: 2112 L: 506 L: 865 D: 857
D: 1598 Q: 682 L: 611 116 TYR S: 7525 T: 8380 Y: 10000 Y: 10000 Y:
9428 Y: 6205 H: 1972 S: 734 F: 3186 T: 577 117 PHE K: 10000 R: 5996
K: 5592 K: 5038 F: 10000 F: 9956 S: 734 Q: 2504 E: 4955 E: 1856 119
ASP N: 3258 N: 4613 N: 9068 R: 2589 D: 4434 D: 842 Q: 1910 S: 665
D: 1614 120 SER R: 9814 Q: 9548 E: 10000 121 SER N: 8165 N: 8910 W:
10000 Q: 1581 Q: 791 122 ASN Q: 9767 R: 7212 Q: 10000 Q: 2768 123
VAL R: 4692 Q: 3725 R: 10000 K: 4955 V: 10000 V: 9965 N: 4665 K:
3427 Q: 2595 E: 2351 E: 1252 R: 999 125 LEU Q: 2247 Q: 2148 Q: 2688
Q: 2618 L: 10000 E: 7583 E: 2234 E: 2024 E: 2252 E: 1838 L: 1122 N:
2103 D: 1558 D: 1815 T: 1591 Q: 540 D: 1840 N: 925 R: 1215 D: 1522
R: 854 N: 828 R: 897 T: 681 S: 553 126 LYS E: 10000 D: 10000 E:
7981 E: 10000 Q: 8882 Q: 7360 Q: 2019 E: 904 R: 2587 127 LYS Q:
10000 Q: 7702 Q: 10000 Q: 7301 T: 8654 Q: 10000 N: 2120 N: 1850 D:
1346 D: 849 128 TYR K: 7798 M: 10000 M: 10000 M: 10000 W: 10000 W:
10000 M: 2139 129 ARG D: 9142 E: 7628 E: 10000 E: 7301 N: 8799 N:
7847 N: 858 D: 1971 R: 2699 D: 1201 Q: 1495 D: 541 134 ARG M: 6832
E: 9900 E: 10000 E: 9997 E: 9996 Q: 5893 E: 3143 Y: 2281 E:
1787
[0174] PDA.RTM. technology calculations were also performed to
identify mutations that are likely to either increase or
substantially eliminate binding to the BMP inhibitor protein
Noggin. At each position in BMP-7 that is within 5 A of at least
one atom in Noggin (see table above), energies were calculated for
alternate residues using a template comprising (1) BMP-7 only, and
(2) BMP-7 bound to Noggin. Preferred substitutions include, but are
not limited to, those listed in the tables below. TABLE-US-00007
TABLE 7 Preferred substitutions to substantially eliminate Noggin
binding Energy Energy Residue Alternate (BMP-7 (BMP-7- number amino
acid only) noggin) .DELTA.(Energy) 55 ILE 5.38 109.30 103.91 55 LEU
4.01 421.61 417.60 55 LYS 0.94 2576.09 2575.15 55 ARG -1.82 1012.10
1013.92 57 MET 4.30 219.47 215.17 57 TYR 7.74 42890.27 42882.53 57
GLU -3.05 119.37 122.42 57 HIS 6.53 2905.10 2898.57 57 HSP 5.72
3035.01 3029.29 57 LYS 0.40 114.98 114.58 57 GLN -4.66 150.57
155.23 57 ARG -3.67 4086.31 4089.97 58 ILE 9.42 322.50 313.09 58
LEU 9.30 84822.06 84812.76 58 MET 8.88 754.69 745.81 58 TYR 4.94
1105.51 1100.57 58 VAL 6.66 226.71 220.05 58 GLU -1.90 199.69
201.59 58 HIS 5.03 1385.50 1380.47 58 HSP 4.31 1951.88 1947.58 58
LYS 3.52 987.81 984.28 58 GLN -3.02 181.68 184.70 58 ARG -3.00
221.94 224.94 59 TYR 9.57 15040.62 15031.05 76 GLU 3.88 210.29
206.41 76 GLN 2.78 304.90 302.12 76 ARG 8.02 9204.62 9196.60 77 GLU
9.73 5019.33 5009.59 77 GLN 8.40 4804.74 4796.34 83 PHE 3.55
2730.52 2726.97 83 TRP 1.99 4103.04 4101.05 83 TYR -3.56 1606.78
1610.35 83 HSP -1.42 110.31 111.73 83 LYS 0.82 275.49 274.67 83 ARG
-4.14 114.90 119.04 86 LEU 6.41 177.93 171.52 86 MET 3.76 182.42
178.66 86 PHE 4.06 510.92 506.87 86 TYR 0.29 486.51 486.22 86 ARG
-1.29 360.80 362.09 87 HIS 4.35 722.22 717.87 87 HSP 9.18 650.44
641.27 113 ILE 9.08 220.54 211.47 113 LEU 4.06 1142.97 1138.91 113
MET 6.10 1203.71 1197.61 113 PHE 5.50 ***** 113 TYR 5.35 ***** 113
GLU -2.87 199.31 202.17 113 HIS -0.15 219.95 220.09 113 HSP 2.62
294.44 291.82 113 LYS 2.39 219.74 217.35 113 GLN -3.68 419.42
423.10 113 ARG -0.22 31582.79 31583.01 115 MET 0.59 433.33 432.74
115 LYS -4.58 104.47 109.05 115 ARG -2.89 629.82 632.71 123 MET
2.17 186.71 184.54 123 TYR 7.81 ***** 123 HIS 6.31 2238.61 2232.29
123 HSP 5.63 11040.76 11035.13 127 ILE 7.26 270.14 262.88 127 VAL
5.22 223.61 218.39 127 HIS 8.31 1012.09 1003.79 127 HSP 8.00
1708.17 1700.17 128 ILE 0.25 401.32 401.07 128 ARG -3.86 124.31
128.17
[0175] TABLE-US-00008 TABLE 8 Preferred substitutions to increase
Noggin binding affinity Energy Energy Residue Alternate (BMP-7
(BMP-7- number amino acid only) noggin) .DELTA.(Energy) 48 MET 8.38
-3.60 -11.99 57 VAL -3.57 -14.50 -10.92 59 MET 7.34 -7.13 -14.47 60
ILE 14.56 -0.05 -14.61 60 LEU 13.50 2.97 -10.52 60 MET 15.34 2.70
-12.64 60 VAL 13.08 0.51 -12.58 74 MET 13.88 1.04 -12.84 76 ILE
13.60 -1.47 -15.07 76 VAL 11.19 -6.60 -17.79 76 ALA 9.97 -3.48
-13.45 77 ALA 13.76 -2.23 -15.99 77 HIS 16.02 2.66 -13.36 77 THR
12.53 -7.56 -20.09 86 VAL 4.41 -10.60 -15.02 113 ALA 2.89 -7.70
-10.58 119 ILE 28.27 18.21 -10.06 119 LEU 29.23 17.25 -11.99 124
VAL -4.30 -14.52 -10.22 125 ILE 247.94 228.79 -19.16 125 MET 10.73
-17.18 -27.91 125 ALA 4.10 -7.22 -11.32 126 MET 8.64 -9.57 -18.21
126 TRP 64.98 35.09 -29.89 126 HIS 49.02 38.84 -10.18 126 HSP 47.75
37.16 -10.59 126 THR 6.31 -5.14 -11.45 127 MET 7.52 -11.44 -18.95
127 ALA 3.79 -6.37 -10.16
[0176] A number of alternate residues were selected for each
variable position. In all cases, the alternate residues are
predicted to be compatible with the structure of BMP-7 dimer. The
alternate residues are predicted to interact with the receptors in
a diverse manner, encompassing competitive inhibitor variants,
receptor specific variants, and high affinity variants. The table
shown below indicates preferred substitutions that were identified
using sequence alignment data, ACE calculations, and PDA.RTM.
technology calculations. Note that "X" indicates a one-residue
deletion. TABLE-US-00009 TABLE 9 BMP-7 variants in Library 1.
Residue wt calculation Library 1.1 Library 1.2 Library 1.3 #
variants 21 LEU expH DKS 3 23 MET expH DKS 3 26 VAL expH DKS 3 36
GLN adtl. surf ENR 3 39 LYS specificity DERST 5 42 GLU adtl. surf
DQRT 4 44 TYR expH AEHKQR 6 48 ARG specificity EKNQ 4 49 ASP adtl.
surf ES 2 52 TRP expH AEKQ 4 53 GLN specificity ADERS H 6 54 ASP
adtl. surf KNRS 4 55 TRP expH AEHKNQ R 7 57 ILE expH AEHKTV D 7 60
GLU specificity KQRST 5 63 ALA adtl. surf EQRS 4 65 TYR
electrostatic DEN 3 70 GLU adtl. surf AQ 2 73 PHE expH AEHQRS D 7
76 ASN specificity ADST 4 77 SER specificity ADKQT 5 78 TYR expH
DGHNST 6 80 ASN glycosylation DQST 4 82 THR glycosylation V 1 83
ASN glycosylation P 1 86 ILE expH EKQT AD 6 88 GLN electrostatic E
1 90 LEU expH EKNQRST 7 93 PHE expH ADEQRST 7 94 ILE expH AEKQRT H
7 95 ASN adtl. surf DKQR 4 97 GLU adtl. surf DKR 3 98 THR adtl.
surf AEKRX 5 108 GLN adtl. surf DKS 3 110 ASN electrostatic DEH 3
111 ALA electrostatic DS 2 115 LEU expH EKT 3 116 TYR expH DEHKST A
7 117 PHE expH ADEKQR H 7 120 SER adtl. surf DERN 4 121 SER adtl.
surf DEKNT 5 122 ASN adtl. surf EQR 3 123 VAL expH ADNRT 5 125 LEU
expH AEKQ Y 5 126 LYS specificity DEQR 4 127 LYS specificity DQST E
5 128 TYR expH DEHKQ 5 129 ARG specificity DES 3 130 ASN
electrostatic D 1 134 ARG specificity EKQS D 5 135 ALA
electrostatic DES 3
[0177] As may easily be appreciated, many of these preferred
substitutions may easily be incorporated into the analogous
positions in other BMPs and TGF-.beta. family members. A sequence
alignment of human BMPs is given in FIG. 3 (SEQ ID NOS:7-12). In
order to identify which substitutions may be incorporated into
BMP-2, BMP-4, BMP-5, BMP-6, and BMP-8, the energy of each of the
above substitutions was calculated in the context of each dimer
structure. Substitutions with similar energies in two different
structures are likely to produce similar effects in the two
proteins. TABLE-US-00010 TABLE 10 Energies of library mutations in
the context of the BMP-2, 4, 5, 6, 7, and 8 structures. BMP-7
Residue substitution BMP2 E(tot) BMP4 E(tot) BMP5 E(tot) BMP6
E(tot) E(tot) BMP8 E(tot) Q 36 GLU 5.6 3.4 6.0 6.4 5.8 5.6 Q 36 ASN
4.2 5.1 2.5 3.0 2.4 2.2 Q 36 ARG 8.0 5.2 5.4 5.7 5.3 5.0 A 37 ASP
-3.0 -3.1 -0.2 1.7 -0.3 -3.4 A 37 GLU -1.8 -2.4 5.0 3.5 2.9 1.3 A
37 HIS 2.3 2.5 10.6 7.1 8.5 5.1 A 37 LYS 2.5 2.7 2.9 1.1 2.7 -0.8 A
37 ARG 1.0 1.1 4.2 2.8 4.1 4.7 K 39 ASP 1.7 1.7 3.6 1.7 2.2 3.9 K
39 GLU 0.7 0.6 -0.7 -2.7 -0.4 0.9 K 39 ARG 2.5 2.5 1.8 0.0 3.2 2.1
K 39 SER -0.2 -0.3 2.0 0.0 1.8 2.9 K 39 THR -0.7 -0.7 1.2 1.2 2.3
1.6 E 42 ASP 4.2 1.3 2.7 3.4 2.7 2.5 E 42 GLN 1.3 0.0 -0.8 1.4 -0.7
0.0 E 42 ARG 1.9 1.3 2.2 4.6 1.9 1.5 E 42 THR 4.4 3.3 4.2 5.0 4.1
3.7 Y 44 ALA 2.6 2.0 7.3 7.3 7.3 8.3 Y 44 GLU -3.5 -4.1 -0.2 -0.2
-0.1 1.3 Y 44 HIS -2.4 -2.6 3.0 2.9 3.0 3.9 Y 44 LYS -2.6 -2.9 3.3
3.4 3.2 5.5 Y 44 GLN -4.4 -4.9 -1.1 -1.1 -1.0 0.4 Y 44 ARG -2.8
-2.9 2.6 2.7 2.6 4.5 R 48 GLU 2.0 2.0 -1.5 -1.7 -1.5 1.2 R 48 LYS
7.0 7.0 2.2 2.9 2.2 7.0 R 48 ASN -0.6 -0.6 -0.1 -0.1 0.1 -1.7 R 48
GLN 0.3 0.4 -0.4 -0.4 -0.4 -0.2 D 49 GLU 4.3 4.3 2.2 2.2 2.1 1.8 D
49 SER 4.3 4.3 -0.7 -0.7 -0.9 -1.0 W 52 ALA -4.1 -4.0 2.7 2.7 2.4
2.1 W 52 GLU -6.4 -6.4 -2.3 -2.3 -2.5 -3.4 W 52 LYS -5.1 -5.0 -2.6
-2.6 -2.8 -5.0 W 52 GLN -5.2 -5.0 -3.5 -3.5 -3.7 -4.8 Q 53 ALA 2.0
1.9 0.1 -1.4 0.1 -3.9 Q 53 ASP -1.2 -1.2 -0.3 -2.0 -0.3 -5.3 Q 53
GLU 0.3 0.3 9.1 -1.5 9.1 -6.8 Q 53 HIS 2.8 2.8 1.4 0.5 1.4 1.6 Q 53
ARG -1.6 -1.5 3.5 0.7 3.3 -3.6 Q 53 SER 0.2 0.2 0.2 -1.8 0.2 -3.4 D
54 LYS 12.3 12.4 4.3 4.4 5.5 6.0 D 54 ASN 4.5 4.4 -1.9 -1.9 0.1
-1.6 D 54 ARG 8.6 8.7 -0.3 -0.3 1.6 1.2 D 54 SER 6.1 6.1 -0.2 -0.2
-0.3 0.1 W 55 ALA 1.9 2.0 1.9 1.9 1.8 2.2 W 55 GLU -2.5 -2.4 -1.8
-1.8 -1.8 -1.7 W 55 HIS 0.6 1.4 4.0 4.0 3.7 4.4 W 55 LYS 0.2 0.3
2.7 2.7 2.6 3.0 W 55 ASN -4.0 -3.9 -3.9 -3.9 -3.9 -3.6 W 55 GLN
-3.7 -3.7 -2.7 -2.7 -2.8 -2.5 W 55 ARG -5.0 -4.8 -3.6 -3.6 -3.7
-3.2 I 57 VAL -3.9 -3.6 -2.7 -3.9 -2.8 -2.2 I 57 ALA 1.1 1.5 -0.5
-1.3 -0.6 -0.7 I 57 ASP -2.8 -2.7 -4.2 -5.3 -4.2 -4.3 I 57 GLU -4.8
-4.5 -6.2 -7.1 -6.1 -5.4 I 57 HIS 2.7 2.9 4.2 2.8 4.3 3.5 I 57 LYS
-1.0 -0.9 -5.8 -6.5 -6.1 -2.0 I 57 THR -7.3 -7.1 -1.2 -2.0 -1.2
-0.7 E 60 LYS 5.3 5.1 5.9 6.1 5.4 6.1 E 60 GLN 0.3 0.2 2.8 2.5 0.7
1.3 E 60 ARG 2.0 1.9 3.6 3.7 1.3 5.1 E 60 SER 2.2 2.1 3.5 2.7 2.9
3.2 E 60 THR 3.2 3.1 5.2 5.1 2.7 5.7 A 63 GLU -2.0 -2.0 -0.9 -0.6
-2.2 -4.5 A 63 GLN -2.7 -2.7 -1.7 -1.5 -3.1 -4.7 A 63 ARG -2.3 -2.3
-1.0 -0.3 -3.4 -1.4 A 63 SER -0.4 -0.4 0.5 1.1 1.4 1.4 Y 65 ASP
-4.1 -6.0 -3.4 -3.5 -3.4 -3.7 Y 65 GLU 21.7 19.5 -3.7 -3.8 -3.9
73.9 Y 65 ASN -0.7 -2.4 -0.3 -0.3 -0.4 -0.7 E 70 ALA 4.3 2.2 2.6
4.6 2.6 1.9 E 70 GLN -0.7 -0.9 0.4 1.1 -0.3 -0.3 A 72 ASP 10.7 -1.8
-1.3 -1.3 -1.1 -1.7 A 72 GLU -0.1 -3.6 -1.3 -1.2 -3.1 -1.5 A 72 HIS
3.7 -0.6 2.9 2.9 2.4 2.6 A 72 LYS 1.9 -2.4 1.1 1.1 0.5 0.8 A 72 ASN
9.0 -0.3 2.3 2.3 2.4 1.9 A 72 ARG -4.4 -2.2 -2.9 -2.9 -3.1 -3.1 A
72 SER 2.0 -1.0 -4.3 -4.3 -4.4 -4.6 F 73 ALA 0.2 0.3 4.0 4.0 6.0
4.0 F 73 ASP -1.9 -1.8 3.4 3.4 4.0 3.6 F 73 GLU -3.2 -3.0 1.1 1.1
1.2 1.3 F 73 HIS -0.7 -0.6 3.7 3.7 5.6 2.7 F 73 GLN -4.0 -3.9 0.3
0.3 0.6 0.6 F 73 ARG -4.7 -4.5 -1.2 -1.2 -0.8 -1.0 F 73 SER -1.2
-1.1 1.8 1.8 1.2 1.8 N 76 ALA 4.1 4.0 6.2 6.2 5.9 4.8 N 76 ASP -0.3
-0.3 1.1 1.1 0.9 0.1 N 76 SER 0.4 0.4 2.5 2.5 2.3 1.1 N 76 THR 8.2
7.8 5.9 5.9 5.4 4.0 S 77 ALA 13.7 13.7 15.1 15.1 15.1 14.7 S 77 ASP
6.3 6.4 7.8 7.8 7.9 7.5 S 77 LYS 18.2 18.2 19.4 19.4 19.3 18.8 S 77
GLN 8.9 9.0 9.8 9.8 9.8 9.3 S 77 THR 12.6 12.8 13.7 13.7 13.6 12.0
Y 78 ASP 3.5 3.7 3.4 3.4 5.0 5.7 Y 78 HIS 11.4 10.4 11.7 11.7 12.5
11.2 Y 78 ASN 2.2 2.4 2.6 2.6 4.0 4.8 Y 78 SER 4.2 4.3 4.5 4.5 5.7
6.3 Y 78 THR 8.1 8.2 8.2 8.2 9.3 9.1 I 86 ALA 1.0 0.9 0.2 0.2 -0.1
-0.4 I 86 ASP -1.1 -1.4 -4.8 -4.8 -4.9 -5.5 I 86 GLU -4.2 -4.1 -6.5
-6.5 -6.5 -6.6 I 86 LYS -0.8 -0.8 -7.0 -7.0 -7.2 -7.7 I 86 GLN -2.7
-2.4 -6.2 -6.2 -6.3 -6.6 I 86 THR 2.2 2.3 -1.0 -1.0 -1.1 -1.4 Q 88
GLU -11.9 -9.9 -10.8 -10.8 -10.9 -9.9 L 90 GLU -7.9 -7.8 -6.4 -6.4
-7.1 -8.4 L 90 LYS -4.4 -4.2 -7.0 -6.9 -9.8 -8.5 L 90 ASN -1.7 -1.6
-3.6 -3.6 -2.1 -1.7 L 90 GLN -6.0 -5.9 -5.4 -5.4 -6.0 -7.0 L 90 ARG
-1.4 -1.2 -4.7 -4.7 -5.6 -5.5 L 90 SER -5.4 -5.4 -2.4 -2.4 -2.6
-2.7 L 90 THR -4.0 -3.9 -1.5 -1.5 -2.6 -3.8 F 93 ALA 1.9 2.1 1.3
1.3 1.2 1.2 F 93 ASP -2.1 -2.0 -2.9 -2.8 -3.0 -3.2 F 93 GLU -4.5
-4.4 0.7 0.8 0.4 0.2 F 93 GLN -4.9 -4.8 -0.6 -0.6 -0.9 -1.1 F 93
ARG -3.4 -3.2 -2.6 -2.6 -3.0 -2.9 F 93 SER -2.9 -2.8 -3.7 -3.7 -3.6
-3.6 F 93 THR 3.9 4.0 6.7 6.7 6.3 6.2 I 94 ALA 1.8 1.9 -1.9 0.5 0.6
-2.4 I 94 GLU -4.9 -4.8 -7.6 -5.2 -5.0 -7.8 I 94 HIS -1.7 -1.6 6.1
-0.2 2.9 5.8 I 94 LYS -5.0 -5.0 -8.4 -5.2 -5.7 -8.4 I 94 GLN -3.7
-3.6 -6.1 -4.0 -3.8 -6.2 I 94 ARG -0.4 -0.2 -5.9 -3.4 -3.5 -4.2 I
94 THR 0.4 0.4 -1.7 0.5 1.0 -1.1 N 95 ASP 0.8 2.8 -4.1 2986.4 -1.7
-2.1 N 95 LYS 3.8 9.2 -1.2 4982.8 3.9 2.7 N 95 GLN 0.6 3.4 -4.9
3497.0 -2.2 0.0 N 95 ARG 2.6 6.3 -2.5 4116.5 3.7 2.4 E 97 ASP 3.2
3.6 6.6 3.3 3.5 6.1 E 97 LYS 3.7 3.9 15.6 12.9 13.1 15.9 E 97 ARG
4.3 4.2 11.4 8.0 8.2 11.1 T 98 ALA 8.5 8.2 0.4 -0.4 -0.4 -0.6 T 98
GLU 4.3 5.6 -5.5 -3.7 -2.8 -4.3 T 98 LYS 9.5 11.1 -5.8 -3.4 -2.4
-2.2 T 98 ARG 6.0 7.9 0.6 3.1 3.5 3.1 A 105 VAL -11.4 -14.8 -4.2
-4.2 0.4 -4.2 Q 108 ASP -1.6 -1.6 -3.3 -3.4 -3.3 -2.2 Q 108 LYS 4.4
4.4 2.8 3.0 2.8 3.3 Q 108 SER -0.6 -0.7 -2.6 -2.6 -2.6 -1.3 N 110
ASP -0.9 -0.7 7.5 7.5 12.8 -0.5 N 110 GLU -0.6 -0.5 -1.6 -1.7 -1.4
0.0 N 110 HIS 10.3 10.8 11.8 11.8 12.4 8.9 A 111 ASP 266.0 263.9
1199.6 1199.6 1155.7 1201.4 A 111 SER 2.5 2.5 1.4 1.4 1.4 3.4 L 115
GLU -7.9 -7.9 -7.8 -7.9 -7.7 -7.8 L 115 LYS -7.2 -7.2 -7.6 -7.5
-7.8 -7.5 L 115 THR -1.3 -2.6 -5.4 -5.4 -5.4 -5.4 Y 116 ASP 22.1
24.7 275.0 274.9 278.8 274.6 Y 116 GLU 76.1 65.5 125.3 125.2 126.5
122.8 Y 116 HIS -9.2 -9.5 -7.5 -7.5 -7.1 -6.7 Y 116 LYS 4301.8
3766.9 198.7 198.7 211.7 203.6 Y 116 SER -2.3 -2.3 -5.6 -5.6 -5.6
-4.3 Y 116 THR -4.8 -4.8 -4.2 -4.3 -4.1 -2.5 F 117 ALA -3.5 -2.5
-0.1 -0.1 -2.0 -2.7 F 117 ASP -5.1 -4.7 -2.4 -2.5 -2.2 -4.7 F 117
GLU -7.2 -7.1 -4.6 -4.7 -4.4 -6.8 F 117 LYS -6.4 -7.6 -2.9 -2.8
-2.7 -5.5 F 117 GLN -8.1 -8.0 -4.7 -4.7 -4.8 -8.0 F 117 ARG -6.5
-5.8 -4.8 -4.7 -4.7 -5.8 D 119 GLU 10.2 12.8 6.8 5.6 6.9 6.7 D 119
ASN 4.5 7.3 6.0 4.9 6.1 6.0 D 119 SER 6.5 9.3 8.0 6.9 8.0 7.9 D 119
THR 12.2 14.3 10.4 10.3 10.5 10.4 S 120 ASP 1.9 1.8 1.5 1.5 1.6 1.0
S 120 GLU 1.1 1.0 3.5 3.5 3.6 2.9 S 120 ASN 0.5 0.3 0.1 0.1 0.2 0.0
S 120 ARG 1.6 1.4 5.3 5.3 5.4 5.0 S 121 ASP 3.2 3.4 3.2 3.2 3.2 2.5
S 121 GLU 4.2 4.4 3.9 3.8 3.9 3.2 S 121 LYS 9.7 9.8 7.9 8.0 7.8 8.1
S 121 ASN 1.8 1.9 1.7 1.7 1.7 1.1 S 121 THR 8.2 8.4 6.5 6.5 6.4 5.7
N 122 GLU -0.3 -0.1 -1.0 -1.1 2.9 -1.1 N 122 GLN -2.0 -1.8 -2.6
-2.6 1.4 -2.6 N 122 ARG -0.8 -0.5 -2.6 -2.9 1.2 -2.6 V 123 ALA 0.7
0.9 1.9 1.9 1.7 1.7 V 123 ASP -1.8 -1.8 -1.0 -1.1 -0.8 -1.2 V 123
ASN -2.9 -2.8 0.1 0.1 0.3 0.0 V 123 ARG -3.3 -2.9 -2.7 -2.6 -2.4
-3.0 V 123 THR -2.6 -2.3 -2.2 -2.2 -2.4 -3.2 L 125 ALA 5.0 5.2 4.7
4.7 4.5 4.7 L 125 GLU -0.8 -0.8 -1.2 -1.3 -1.3 -1.2 L 125 LYS 4.2
4.2 3.6 3.7 3.4 3.7 L 125 GLN -1.8 -1.7 -2.2 -2.2 -2.3 -2.2 K 126
ASP -1.6 -3.4 -5.5 -5.5 -5.5 -5.2 K 126 GLU -1.4 -3.0 -3.3 -3.4
-5.4 -2.7 K 126 GLN -2.4 -4.3 -1.4 -1.4 -3.6 -3.3 K 126 ARG -2.7
-5.7 0.5 0.5 -0.7 9.9 K 127 ASP 5.9 4.0 0.7 0.7 0.6 0.9 K 127 GLN
2.0 0.2 -2.5 -2.5 -3.1 -2.3 K 127 SER 3.0 1.2 0.1 0.1 -0.1 0.1 K
127 THR 5.6 3.7 1.5 1.5 -0.5 1.7 Y 128 ASP -6.4 -6.6 -5.5 -5.5 -5.6
-5.3 Y 128 GLU -3.3 -3.0 -7.4 -7.5 -7.6 -8.9 Y 128 HIS -10.0 -9.8
-4.3 -4.3 -4.4 -7.3 Y 128 LYS 8.1 10.0 -8.7 -8.6 -8.8 -5.9 Y 128
GLN 3.6 3.6 -5.7 -5.7 -5.8 -4.6 R 129 ASP -0.5 -0.5 0.1 0.0 0.1 0.1
R 129 GLU -0.3 1.6 -2.0 -2.0 -2.0 -1.9 R 129 SER -0.4 1.5 1.5 1.5
1.5 1.5 N 130 ASP -6.6 -4.6 -2.8 -2.8 -2.6 -2.7 R 134 GLU -6.9 -6.9
-7.1 -7.1 -6.9 -6.9 R 134 LYS 12.3 18.6 -4.3 -4.1 -2.2 -4.1 R 134
GLN -4.3 -4.2 -7.5 -7.4 -7.4 -6.6 R 134 SER -2.4 -2.4 -3.8 -3.8
-3.9 -3.2 A 135 ASP 1.6 1.2 -5.1 -5.1 -5.9 -1.7 A 135 GLU 2.2 1.2
-4.8 -4.8 -5.9 -2.6 A 135 SER 3.7 2.4 -4.0 -4.0 -5.2 -2.1 H 139 ARG
-4.4 -4.4 -0.6 -0.6 -2.6 -0.6
[0178] Correlation coefficients (R.sup.2) for energies calculated
using the BMP-2 template versus other BMP templates are as follows:
BMP2 vs BMP4=0.93, BMP2 vs BMP5=0.52, BMP2 vs BMP6=0.44, BMP2 vs
BMP-7=0.64, and BMP2 vs BMP8=0.52. Correlation coefficients
(R.sup.2) for energies calculated using the BMP-7 template versus
other BMP templates are as follows: BMP-7 vs BMP2=0.54, BMP-7 vs
BMP4=0.54, BMP-7 vs BMP5=0.95, BMP-7 vs BMP6=0.71, and BMP-7 vs
BMP8=0.76. These trends correlate with the sequence similarity of
the pair. Modifications to BMP-7 have substantially similar effects
on other BMPs if the energy difference between the modification in
the BMP-7 template and another BMP template are less than 1
kcal/mol, or if both energies are higher than 50 kcal/mol. The
following BMP-7 modifications have substantially similar effects in
BMP-2: Q36E, A37K, K39D, E42R, E42T, R48N, R48Q, Q53D, Q53S, W55A,
W55E, W55N, W55Q, E60K, E60Q, E60R, E60S, E60T, A63E, A63Q, Y65D,
Y65N, E70Q, S77Q, S77T, Q88E, L90E, L90N, L90Q, F93A, F93D, F93R,
F93S, I94E, I94K, I94Q, I94T, N95K, E97D, N110E, A111D, L115E,
L115K, Y116D, Y116E, Y116K, Y116T, S120D, S120N, S121D, S121E,
S121N, V123A, V123D, V123R, V123T, L125A, L125E, L125K, L125Q,
Y128D, R129D, and R134E. The following BMP-7 modifications have
substantially similar effects in BMP-4: Q36R, A37K, K39D, K39E,
E42Q, E42R, E42T, R48N, R48Q, Q53D, Q53S, W55A, W55E, W55N, W55Q,
157V, E60K, E60Q, E60R, E60S, E60T, A63E, A63Q, E70A, E70Q, A72D,
A72E, A72R, S77Q, S77T, Q88E, L90E, L90N, L90Q, F93A, F93D, F93R,
F93S, I94E, I94K, I94Q, I94T, E97D, N110E, A111D, L115E, L115K,
Y116D, Y116E, Y116K, Y116T, F117A, S120D, S120N, S121D, S121E,
S121N, V123A, V123D, V123R, V123T, L125A, L125E, L125K, L125Q,
K126Q, Y128D, R129D, R129S, and R134E. The following BMP-7
modifications have substantially similar effects in BMP-5: Q36E,
Q36N, Q36R, A37D, A37K, A37R, K39E, K39S, E42D, E42Q, E42R, E42T,
Y44A, Y44E, Y44H, Y44K, Y44Q, Y44R, R48E, R48K, R48N, R48Q, D49E,
D49S, W52A, W52E, W52K, W52Q, Q53A, Q53D, Q53E, Q53H, Q53R, Q53S,
D54S, W55A, W55E, W55H, W55K, W55N, W55Q, W55R, I57V, I57A, I57D,
I57E, I57H, I57K, I57T, E60K, E60S, A63S, Y65D, Y65E, Y65N, E70A,
E70Q, A72D, A72H, A72K, A72N, A72R, A72S, F73D, F73E, F73Q, F73R,
F73S, N76A, N76D, N76S, N76T, S77A, S77D, S77K, S77Q, S77T, Y78H,
I86A, I86D, I86E, I86K, I86T, I86T, Q88E, L90E, L90Q, L90R, L90S,
F93A, F93D, F93E, F93Q, F93R, F93S, F93T, T98A, Q108D, Q108K,
Q108S, N110E, N110H, A111D, A111S, L115E, L115K, L115T, Y116D,
Y116E, Y116H, Y116K, Y116S, Y116T, F117D, F117E, F117K, F117Q,
F117R, D119E, D119N, D119S, D119T, S120D, S120E, S120N, S120R,
S121D, S121E, S121K, S121N, S121T, V123A, V123D, V123N, V123R,
V123T, L125A, L125E, L125K, L125Q, K126D, K127D, K127Q, K127S,
Y128D, Y128E, Y128H, Y128K, Y128Q, R129D, R129E, R129S, N130D,
R134E, R134Q, R134S, and A135D. The following BMP-7 modifications
have substantially similar effects in BMP-6: Q36E, Q36N, Q36R,
A37E, K39D, E42D, E42T, Y44A, Y44E, Y44H, Y44K, Y44Q, Y44R, R48E,
R48K, R48N, R48Q, D49E, D49S, W52A, W52E, W52K, W52Q, Q53H, D54S,
W55A, W55E, W55H, W55K, W55N, W55Q, W55R, I57A, I57E, I57K, I57T,
E60K, E60S, Y65D, Y65E, Y65N, A72D, A72H, A72K, A72N, A72R, A72S,
F73D, F73E, F73Q, F73R, F73S, N76A, N76D, N76S, N76T, S77A, S77D,
S77K, S77Q, S77T, Y78H, I86A, I86D, I86E, I86K, I86T, I86T, Q88E,
L90E, L90Q, L90R, L90S, F93A, F93D, F93E, F93Q, F93R, F93S, F93T,
I94A, I94E, I94K, I94Q, I94R, I94T, E97D, E97K, E97R, T98A, T98E,
T98K, T98R, Q108D, Q108K, Q108S, N110E, N110H, A111D, A111S, L115E,
L115K, L115T, Y116D, Y116E, Y116H, Y116K, Y116S, Y116T, F117D,
F117E, F117K, F117Q, F117R, D119T, S120D, S120E, S120N, S120R,
S121D, S121E, S121K, S121N, S121T, V123A, V123D, V123N, V123R,
V123T, L125A, L125E, L125K, L125Q, K126D, K127D, K127Q, K127S,
Y128D, Y128E, Y128H, Y128K, Y128Q, R129D, R129E, R129S, N130D,
R134E, R134Q, R134S, and A135D. The following BMP-7 modifications
have substantially similar effects in BMP-8: Q36E, Q36N, Q36R,
A37R, K39T, E42D, E42Q, E42R, E42T, Y44A, Y44H, R48Q, D49E, D49S,
W52A, W52E, Q53H, D54K, D54R, D54S, W55A, W55E, W55H, W55K, W55N,
W55Q, W55R, 157V, 157A, 157D, 157E, 157H, 157T, E60K, E60Q, E60S,
A63S, Y65D, Y65N, E70A, E70Q, A72D, A72H, A72K, A72N, A72R, A72S,
F73D, F73E, F73Q, F73R, F73S, N76D, S77A, S77D, S77K, S77Q, Y78D,
Y78N, Y78S, Y78T, I86A, I86D, I86E, I86K, I86T, I86T, Q88E, L90N,
L90Q, L90R, L90S, F93A, F93D, F93E, F93Q, F93R, F93S, F93T, I94R,
N95D, T98A, T98K, T98R, Q108K, A111D, L115E, L115K, L115T, Y116D,
Y116E, Y116H, Y116K, F117A, D119E, D119N, D119S, D119T, S120D,
S120E, S120N, S120R, S121D, S121E, S121K, S121N, S121T, V123A,
V123D, V123N, V123R, V123T, L125A, L125E, L125K, L125Q, K126D,
K126Q, K127D, K127Q, K127S, Y128D, R129D, R129E, R129S, N130D,
R134E, R134Q, and R134S.
[0179] ACE calculations were also performed to assess the
similarity of the structural environment at each variable position
in BMP-7 vs. BMP-2, BMP-4, BMP-5, BMP-6, and BMP-8. At positions
with an ACE similarity score of 0.4 or higher in the table below,
mutations will have similar effects in BMP-7 vs. the other BMP. ACE
similarity scores between 0.6 and 0.8 indicate that the effects of
mutations are highly likely to have similar effects, and ACE
similarity scores greater than 0.8 indicate that the effects of
mutations should be substantially identical. TABLE-US-00011 TABLE
11 ACE similarity scores for BMP-7 versus selected additional human
TGF-.beta. proteins BMP- BMP- BMP-2 BMP-3 3B BMP-4 BMP-5 BMP-6
BMP-8 BMP-9 10 36 0.58 0.11 0.14 0.23 0.85 0.81 0.60 0.10 0.10 37
0.12 0.02 0.02 0.07 0.72 0.27 0.30 0.02 0.05 39 0.35 0.03 0.04 0.24
0.75 0.67 0.38 0.02 0.02 42 0.30 0.37 0.37 0.33 0.56 0.27 0.69 0.36
0.36 44 0.19 0.04 0.04 0.22 0.95 0.82 0.59 0.04 0.21 48 0.17 0.17
0.17 0.17 1.00 0.74 0.26 0.19 0.16 49 0.11 0.09 0.09 0.12 0.56 0.50
0.40 0.07 0.07 52 0.37 0.32 0.34 0.37 0.99 0.98 0.46 0.41 0.39 53
0.28 0.11 0.11 0.28 1.00 0.49 0.48 0.09 0.12 54 0.29 0.30 0.29 0.29
1.00 0.96 0.19 0.29 0.30 55 0.59 0.30 0.26 0.58 1.00 0.99 0.52 0.26
0.17 57 0.20 0.45 0.11 0.20 1.00 0.96 0.43 0.05 0.08 60 0.48 0.18
0.18 0.48 1.00 0.74 0.65 0.21 0.61 63 0.19 0.04 0.03 0.21 0.35 0.10
0.81 0.06 0.31 65 0.04 0.01 0.01 0.05 0.96 0.99 0.20 0.03 0.02 70
0.04 0.02 0.02 0.04 0.38 0.12 0.22 0.03 0.02 72 0.23 0.01 0.01 0.08
0.93 0.80 0.23 0.03 0.01 73 0.18 0.02 0.02 0.17 0.41 0.41 0.12 0.18
0.05 76 0.21 0.12 0.11 0.17 0.58 0.58 0.50 0.10 0.15 77 0.26 0.12
0.12 0.26 0.76 0.76 0.37 0.12 0.16 78 0.34 0.13 0.13 0.28 0.77 0.75
0.61 0.14 0.17 80 0.09 0.01 0.01 0.09 0.32 0.46 0.37 0.04 0.07 82
0.02 0.01 0.01 0.01 0.99 0.99 0.68 0.00 0.00 83 0.41 0.14 0.14 0.36
1.00 1.00 0.44 0.26 0.25 86 0.36 0.09 0.09 0.36 0.97 0.97 0.39 0.10
0.04 88 0.04 0.15 0.15 0.04 0.82 0.82 0.05 0.01 0.00 90 0.18 0.11
0.11 0.18 0.33 0.33 0.18 0.11 0.04 93 0.37 0.39 0.39 0.38 0.59 0.63
0.54 0.32 0.14 94 0.11 0.07 0.07 0.12 0.18 0.34 0.22 0.15 0.16 95
0.00 0.01 0.01 0.00 0.05 0.11 0.05 0.02 0.00 97 0.02 0.05 0.05 0.02
0.06 0.59 0.15 0.06 0.02 98 0.20 0.02 0.02 0.20 0.05 0.88 0.11 0.02
0.07 105 0.03 0.01 0.01 0.03 0.95 0.98 0.69 0.03 0.03 108 0.09 0.03
0.02 0.09 0.37 0.98 0.45 0.05 0.04 110 0.11 0.03 0.01 0.10 0.69
0.70 0.30 0.01 0.02 111 0.02 0.00 0.00 0.01 1.00 1.00 0.23 0.00
0.03 115 0.45 0.39 0.30 0.45 1.00 0.99 0.82 0.08 0.07 116 0.38 0.65
0.55 0.38 1.00 0.98 0.38 0.09 0.04 117 0.36 0.43 0.37 0.35 1.00
0.98 0.61 0.20 0.14 119 0.53 0.58 0.54 0.37 1.00 0.69 0.89 0.38
0.41 120 0.07 0.37 0.32 0.07 1.00 1.00 0.33 0.04 0.01 121 0.13 0.62
0.25 0.10 1.00 0.72 0.53 0.05 0.07 122 0.21 0.22 0.18 0.09 1.00
0.42 0.62 0.08 0.07 123 0.16 0.64 0.21 0.15 1.00 0.99 0.52 0.07
0.09 125 0.69 0.62 0.27 0.69 1.00 1.00 0.84 0.50 0.43 126 0.60 0.33
0.29 0.60 1.00 1.00 0.28 0.46 0.38 127 0.37 0.07 0.03 0.37 1.00
1.00 0.61 0.04 0.03 128 0.55 0.38 0.38 0.55 1.00 1.00 0.30 0.07
0.05 129 0.10 0.05 0.06 0.09 0.99 0.99 0.61 0.01 0.03 130 0.26 0.02
0.03 0.26 0.98 0.98 0.77 0.06 0.07 134 0.07 0.05 0.12 0.07 0.36
0.38 0.12 0.04 0.03 135 0.10 0.00 0.00 0.10 0.23 0.22 0.10 0.04
0.03 139 0.19 0.01 0.00 0.18 0.94 0.97 0.80 0.10 0.10 BMP- TGF-
TGF- TGF- TGF- 15 GDF-1 GDF-3 GDF-5 GDF-8 GDF-9 .beta.1 .beta.2
.beta.3 .beta.4 36 0.14 0.42 0.41 0.10 0.36 0.24 0.10 0.10 0.10
0.09 37 0.02 0.02 0.03 0.03 0.01 0.02 0.01 0.01 0.01 0.00 39 0.19
0.07 0.23 0.04 0.06 0.19 0.02 0.02 0.03 0.02 42 0.11 0.11 0.15 0.21
0.11 0.09 0.09 0.09 0.09 0.34 44 0.03 0.29 0.19 0.04 0.03 0.03 0.18
0.17 0.17 0.12 48 0.24 0.28 0.21 0.15 0.16 0.22 0.21 0.21 0.21 0.15
49 0.40 0.27 0.20 0.12 0.06 0.12 0.09 0.15 0.14 0.06 52 0.62 0.26
0.49 0.42 0.08 0.15 0.36 0.37 0.34 0.01 53 0.18 0.14 0.17 0.45 0.03
0.01 0.07 0.04 0.06 0.01 54 0.30 0.31 0.30 0.30 0.30 0.09 0.39 0.38
0.38 0.12 55 0.10 0.14 0.16 0.45 0.08 0.11 0.07 0.07 0.07 0.12 57
0.12 0.73 0.10 0.17 0.62 0.11 0.01 0.03 0.03 0.00 60 0.32 0.90 0.72
0.27 0.20 0.22 0.51 0.41 0.50 0.31 63 0.00 0.03 0.02 0.06 0.01 0.00
0.05 0.04 0.04 0.18 65 0.01 0.02 0.05 0.03 0.02 0.01 0.02 0.01 0.01
0.02 70 0.02 0.05 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.00 72 0.00
0.00 0.02 0.00 0.00 0.00 0.00 0.00 0.00 0.00 73 0.02 0.04 0.02 0.12
0.00 0.01 0.00 0.00 0.00 0.00 76 0.01 0.05 0.02 0.25 0.01 0.01 0.00
0.00 0.00 0.05 77 0.22 0.11 0.24 0.20 0.07 0.13 0.11 0.11 0.11 0.14
78 0.23 0.11 0.24 0.21 0.08 0.10 0.08 0.12 0.10 0.14 80 0.00 0.01
0.04 0.11 0.03 0.00 0.00 0.00 0.00 0.01 82 0.00 0.01 0.00 0.01 0.00
0.00 0.00 0.00 0.00 0.00 83 0.13 0.10 0.08 0.26 0.01 0.08 0.02 0.02
0.03 0.01 86 0.09 0.04 0.11 0.21 0.00 0.01 0.00 0.00 0.00 0.00 88
0.07 0.02 0.00 0.01 0.00 0.04 0.00 0.00 0.00 0.00 90 0.05 0.02 0.10
0.09 0.01 0.01 0.02 0.06 0.04 0.00 93 0.14 0.22 0.62 0.40 0.34 0.06
0.15 0.43 0.30 0.07 94 0.07 0.07 0.11 0.09 0.12 0.04 0.08 0.10 0.10
0.01 95 0.01 0.02 0.01 0.14 0.02 0.01 0.01 0.11 0.09 0.00 97 0.01
0.08 0.18 0.15 0.50 0.01 0.43 0.44 0.44 0.01 98 0.01 0.01 0.05 0.03
0.11 0.01 0.03 0.06 0.05 0.00 105 0.00 0.02 0.00 0.03 0.02 0.00
0.00 0.00 0.00 0.00 108 0.02 0.02 0.05 0.10 0.03 0.01 0.05 0.03
0.04 0.03 110 0.03 0.03 0.03 0.05 0.02 0.02 0.14 0.06 0.08 0.01 111
0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 115 0.15 0.73
0.55 0.23 0.22 0.09 0.00 0.00 0.00 0.01 116 0.22 0.42 0.31 0.46
0.07 0.10 0.01 0.01 0.01 0.01 117 0.23 0.43 0.51 0.51 0.29 0.16
0.01 0.01 0.01 0.04 119 0.34 0.84 0.57 0.58 0.29 0.29 0.16 0.19
0.17 0.21 120 0.02 0.33 0.32 0.31 0.01 0.02 0.00 0.00 0.00 0.01 121
0.09 0.83 0.16 0.24 0.17 0.08 0.04 0.04 0.04 0.05 122 0.04 0.52
0.21 0.21 0.03 0.02 0.00 0.00 0.00 0.01 123 0.11 0.67 0.17 0.21
0.43 0.06 0.01 0.03 0.03 0.00 125 0.62 0.67 0.65 0.71 0.32 0.47
0.05 0.14 0.14 0.01 126 0.15 0.39 0.61 0.31 0.07 0.08 0.03 0.03
0.03 0.01 127 0.05 0.05 0.05 0.05 0.01 0.04 0.01 0.02 0.01 0.01 128
0.45 0.25 0.20 0.49 0.03 0.15 0.05 0.06 0.06 0.02 129 0.01 0.02
0.01 0.02 0.02 0.01 0.03 0.03 0.03 0.01 130 0.01 0.03 0.04 0.14
0.01 0.00 0.01 0.01 0.01 0.00 134 0.01 0.03 0.04 0.07 0.04 0.02
0.02 0.02 0.02 0.01 135 0.00 0.00 0.05 0.03 0.02 0.00 0.00 0.00
0.00 0.01 139 0.00 0.01 0.03 0.11 0.02 0.00 0.00 0.00 0.00 0.00
[0180] Based on the above ACE analysis, BMP-7 variants at the
following positions are transferable to BMP-2: 36, 55, 60, 83, 115,
119, 125, 126, and 128. BMP-7 variants at the following positions
are transferable to BMP-3: 57, 116, 117, 119, 121, 123, and 125.
BMP-7 variants at the following positions are transferable to
BMP-3b: 116 and 119. BMP-7 variants at the following positions are
transferable to BMP-4: 55, 60, 115, 125, 126, and 128. BMP-7
variants at the following positions are transferable to BMP-5: 36,
37, 39, 42, 44, 48, 49, 52, 53, 54, 55, 57, 60, 65, 72, 73, 76, 77,
78, 82, 83, 86, 88, 93, 105, 110, 111, 115, 116, 117, 119, 120,
121, 122, 123, 125, 126, 127, 128, 129, 130, and 139. BMP-7
variants at the following positions are transferable to BMP-6: 36,
39, 44, 48, 49, 52, 53, 54, 55, 57, 60, 65, 72, 73, 76, 77, 78, 80,
82, 83, 86, 88, 93, 97, 98, 105, 108, 110, 111, 115, 116, 117, 119,
120, 121, 122, 123, 125, 126, 127, 128, 129, 130, and 139. BMP-7
variants at the following positions are transferable to BMP-9: 36,
42, 44, 49, 52, 53, 55, 57, 60, 63, 75, 78, 82, 83, 93, 105, 108,
115, 117, 119, 121, 122, 123, 125, 127, 129, 130, and 139. BMP-7
variants at the following positions are transferable to BMP-9: 52,
125, and 126. BMP-7 variants at the following positions are
transferable to BMP-10: 60, 119, and 125. BMP-7 variants at the
following positions are transferable to BMP-15: 49, 52, 125, and
128. BMP-7 variants at the following positions are transferable to
GDF-1: 36, 57, 60, 115, 116, 117, 119, 121, 123, and 125. BMP-7
variants at the following positions are transferable to GDF-3: 36,
52, 60, 93, 115, 117, 119, 125, and 126. BMP-7 variants at the
following positions are transferable to GDF-5: 52, 53, 55, 93, 116,
117, 119, 125, and 128. BMP-7 variants at the following positions
are transferable to GDF-8: 57, 97, and 123. BMP-7 variants at the
following positions are transferable to GDF-9: 125. BMP-7 variants
at the following positions are transferable to TGF-.beta.1: 60 and
97. BMP-7 variants at the following positions are transferable to
TGF-.beta.2: 60, 93 and 97. BMP-7 variants at the following
positions are transferable to TGF-.beta.3: 60 and 97.
Example 6
Generation of DNA Encoding the Parent BMP-7
[0181] A number of constructs for the expression of wild type human
BMP-7 were made and tested: (1) native BMP-7 Image clone in pSport6
vector (obtained from ATCC), (2) FLAG-tagged BMP-7, with G4S linker
and FLAG tag located just after the RXXR (SEQ ID NO:87) cleavage
site in pCMVTnT vector (Promega), (3) FLAG-tagged BMP-7 with a
perfect Kozak sequence (MAV rather than MHV) in pCMVTnT vector, (4)
FLAG-tagged BMP-7 using native BMP-2 Kozak sequence, signal
sequence, and pro-domain in pCMVTnT vector, (5) FLAG-tagged BMP-7
using native MIC-1 Kozak sequence, signal sequence, and pro-domain
in pCMVTnT vector, and (6) native BMP-7 Image clone in pSport6
vector (obtained from ATCC) with 6.times.His tag at the N-terminus
of the pro-domain. The untagged native Image clone in the pSport6
vector was found to produce the highest expression yields and was
used as the template for variant construction.
[0182] Additional constructs were prepared for yeast expression:
(1) pYES vector negative control, (2) BMP-7 mature domain in pYES
vector, (3) E-BMP-7 mature domain in pYES vector, (4) AEAE-BMP-7
mature domain in pYES vector, (5) full length BMP-7 in pYES vector,
and (6) REKR-full length BMP-7 in pYES vector.
Example 7
Generation of DNA Encoding BMP Variants
[0183] Constructs for the BMP-7 variants shown in the above table
were prepared by site directed mutagenesis. The pSport6 Image clone
(ATCC) was used as a template for all of the variants. DNA
corresponding to each desired construct was prepared using Qiagen
Miniprep or Maxiprep kits. As some of the BMP-7 variants were
constructed using degenerate oligos, variants in addition to those
explicitly included in Library 1 above were generated. Such
variants include the following: L21E, L21G, L21N, L21R, M23G, M23N,
M23R, V26E, V26G, V26N, K39A, K39G, K39N, Y44D, Y44G, Y44N, Y44P,
Y44S, Y44T, R48D, R48H, W52P, W52T, Q53G, Q53K, Q53T, W55P, W55T,
157L, 157P, 157Q, E60H, E60N, E60P, F73G, F73K, F73N, F73T, N76Y,
S77E, S77H, S77N, S77P, Y78P, Y78R, 186P, L90G, L90H, L90P, F93G,
F93H, F93P, L115A, L115Q, Y116A, Y116Q, F117S, F117Y, V123G, I125P,
I125T, K126G, K127A, K127H, K127N, K127P, K127Y, R129K, R129N,
R134L, and R134P. Furthermore, silent mutations (that is, changes
in DNA codon sequence that do not change the corresponding amino
acid sequence) were introduced at I57, N76, Y116, V123, and
R134.
Example 8
In Vitro Translation of Wild Type BMP-7
[0184] The following constructs were generated and used for in
vitro translation experiments: (1) full length BMP-7 with FLAG tag
at N-terminus of mature domain, (2) full length MIC-1 with FLAG tag
at the N-terminus of the mature domain, (3) BMP-7 pro-domain with
N-terminal FLAG tag, (4) MIC-1 pro-domain with N-terminal FLAG tag,
(5) BMP-7 mature domain with N-terminal FLAG tag, (6) MIC-1 mature
domain with N-terminal FLAG tag, (7) full length BMP-7 with
C-terminal FLAG tag, (8) full length MIC-1 with C-terminal FLAG
tag, (9) BMP-7 pro-domain with C-terminal FLAG tag, (10) MIC-1
pro-domain with C-terminal FLAG tag, (11) BMP-7 mature domain with
C-terminal FLAG tag, and (12) MIC-1 mature domain with C-terminal
FLAG tag. The 5' end of each construct had a spacer, T7 promoter,
globin UTR, and optimized ribosomal binding site preceeding the
gene. Luciferase was used as a positive control. A coupled
transcription/translation off the PCR product was used (TnT,
Promega). Protein expression comparable to luciferase was obtained
for all of the BMP-7 constructs other than (11) above.
Example 9
Expression of Wild Type and Variant BMP-7
[0185] The following small-scale expression protocol was used for
initial library screening. 293T cells were plated into 6-well
dishes (1-5.times.10.sup.5 cells/mL in 4 mL DMEM 10% FBS). The next
day, the cells were transfected using 5 ug DNA/well. As an internal
control for transformation efficiency, several mini-preps of the
native Image clone were used. After three days, the conditioned
media was harvested and screened. Expression yields were determined
using ELISA (all variants, not corrected for changes in antibody
binding affinity, using R&D Systems ELISA Duoset Cat#DY354) and
Western blotting (selected variants). TABLE-US-00012 TABLE 12
Expression yields of Library 1 variants in 293T cells Fold change
ELISA relative to Western Variant concentration image blot band
name (ng/mL) clone intensity L21D 999 1.34 L21E 332 0.45 L21G 4917
0.78 ++ L21K 495 0.50 L21N 414 2.97 - L21R 682 0.92 M23D 565 0.56 +
M23G 4 0.03 +++ M23N 600 0.81 + M23R 3 0.02 - M23S 15 0.11 - V26D
747 0.75 + V26E 1021 1.37 + V26G 605 0.81 - V26K 0 0.00 - V26N 976
1.31 + V26S 6 0.01 - Q36E 7107 1.13 ++ Q36N 1 0.00 - K39A 22724
3.61 + K39D 19 0.01 K39E 1 0.00 K39G 1 0.00 K39N 69 0.04 K39R 8376
1.33 +++ K39S 23063 3.67 + K39T 19 0.01 E42D 7307 1.16 +++ E42Q
6702 1.07 +++ E42R 583 0.58 - E42T 449 0.45 - Y44A 259 0.04 - Y44D
1 0.00 Y44E 42 0.04 - Y44G 0 0.00 Y44H 1 0.01 Y44K 2 0.00 - Y44N 14
0.10 - Y44P 0 0.00 Y44Q 19 0.02 - Y44S 1 0.00 - Y44T 21 0.15 R48D 1
0.00 R48E 74 0.04 R48H 6063 0.96 - R48N 7100 1.13 - R48Q 743 0.42
D49S 24 0.02 - W52A 38 0.27 W52E 1 0.00 W52K 0 0.00 W52P 407 0.55
W52Q 6 0.01 - W52T 5 0.03 Q53A 251 0.04 - Q53D 6876 3.87 ++ Q53E
1237 0.70 Q53G 83 0.05 + Q53H 645 0.29 ++ Q53K 6413 1.02 + Q53R 149
0.08 Q53S 8530 1.36 +++ Q53T 13237 2.10 ++ D54K 10 0.01 - D54N 458
0.46 - D54S 38 0.01 - W55A 26 0.04 W55E 2 0.00 W55H 6 0.01 W55K 10
0.07 W55N 11 0.08 W55P 0 0.00 W55Q 10 0.07 W55R 48 0.34 - W55T 0
0.00 I57A 47 0.01 - I57D 0 0.00 I57E 0 0.00 I57H 8 0.06 I57I 526
0.71 - I57K 9 0.06 I57L 16106 2.56 0.63 I57P 0 0.00 I57Q 0 0.00
I57T 1 0.00 - I57V 6 0.01 E60H 129 0.07 E60K 755 0.76 ++ E60N 5
0.00 E60P 145 0.08 E60Q 3172 3.17 + E60R 2069 0.33 + E60S 97 0.05
E60T 410 0.41 - A63E 4068 4.07 + A63Q 8577 8.58 ++ A63R 3183 3.18 +
A63S 8448 8.45 ++ Y65D 18743 18.74 +++ Y65E 382 0.38 - Y65N 11678
11.68 ++ E70A 501 0.50 - E70Q 679 0.68 - A72D 5462 5.46 + A72E 3822
3.82 - A72H 3308 3.31 + A72K 3863 3.86 + A72N 1927 0.88 + A72R 491
0.49 - A72S 4784 2.17 ++ F73A 153 1.10 F73D 27 0.19 F73E 138 0.99
F73G 173 1.24 F73H 5783 5.78 ++ F73K 25 0.18 F73N 321 0.43 F73Q 75
0.54 F73S 5159 0.82 ++ F73T 193 1.38 - N76A 5570 3.14 N76D 7534
4.24 N76S 8816 4.97 N76T 4576 2.58 N76Y 814 0.46 S77A 1194 0.67
S77D 1 0.00 S77E 46 0.03 S77H 49 0.03 S77K 1 0.00 S77N 1 0.00 S77P
1302 0.73 S77Q 645 0.64 - S77T 1048 0.59 Y78D 517 3.71 - Y78G 2
0.00 Y78H 10102 1.61 + Y78N 5795 5.80 - Y78P 1249 1.68 Y78R 264
1.89 - Y78S 9648 1.53 ++ Y78T 714 5.12 - I86A 5635 5.64 ++ I86D
3480 3.48 ++ I86E 2556 3.43 I86K 2214 2.97 I86P 5 0.01 I86Q 3008
4.04 I86T 532 0.53 + Q88E 47 0.05 - L90E 235 0.32 L90H 62 0.08 L90H
62 0.08 L90K 3 0.00 + L90N 200 0.09 +++ L90P 109 0.15 L90R 356 0.48
L90S 5463 0.87 +++ L90T 647 0.87 F93A 12 0.02 F93D 2346 0.37 ++
F93E 646 0.87 ++ F93G 24 0.03 ++ F93H 16307 2.59 +++ F93P 15 0.01
+++ F93Q 279 0.13 +++ F93R 38 0.02 ++ F93S 2836 1.29 ++ F93T 287
0.39 + I94A 2 0.00 +++ I94E 1 0.00 - I94H 70 0.03 - I94K 0 0.00 -
I94P 0 0.00 I94Q 854 1.15 I94R 0 0.00 + I94T 10 0.01 - N95D 18 0.02
- N95K 2 0.00 - N95Q 53 0.05 - N95R 1 0.00 - E97D 4784 2.17 - E97K
2936 1.33 - E97R 2113 0.96 - T98A 4208 1.91 - T98E 3064 1.39 T98K
5464 2.48 - T98X 927 0.93 - A105V 440 0.44 - Q108D 4052 1.84 -
Q108K 1 0.00 - Q108S 3529 1.60 - N110D 8591 3.91 - N110E 3020 1.37
- N110H 331 0.33 - A111D 2317 0.37 ++ A111S 4358 4.36 - L115A 24
0.03 L115E 1 0.00 - L115K 1 0.00 - L115Q 1 0.00 - L115T 0 0.00
Y116A 0 0.00 Y116D 1 0.00 - Y116E 0 0.00 Y116H 16724 2.66 + Y116K 0
0.00 Y116Q 0 0.00 Y116S 34 0.01 - Y116T 2 0.00 - Y116Y 1922 2.58
F117A 2 0.00 - F117D 0 0.00 F117E 2407 0.38 - F117H 18720 2.98 ++
F117K 329 0.44 F117Q 3730 0.59 + F117R 604 0.81 F117S 42 0.06 F117Y
1682 2.26 D119E 9669 1.54 ++ D119N 2870 0.46 - D119S 4824 0.77 +
D119T 2022 0.32 - S120D 21370 3.40 +++ S120E 9259 1.47 ++ S120N
11035 1.75 ++ S120R 10544 1.68 ++ S121D 9202 1.46 ++ S121E 6098
0.97 +
S121K 4793 0.76 - S121N 5161 0.82 - S121T 2204 0.35 - N122E 1 0.00
- N122Q 6 0.00 - N122R 63 0.01 - V123A 0 0.00 V123D 1 0.00 - V123G
305 0.41 V123N 0 0.00 V123R 1 0.00 - V123T 1 0.00 V123V 6 0.01
L125A 1 0.00 L125E 0 0.00 L125K 1 0.00 - L125P 0 0.00 L125Q 4 0.00
- L125T 0 0.00 L125Y 3306 0.53 - K126D 4393 0.70 - K126E 24 0.01
K126G 55 0.03 K126Q 145 0.08 K126R 10478 1.67 + K127A 1 0.00 K127D
1 0.00 K127E 3974 0.63 ++ K127H 1 0.00 K127N 1 0.00 K127P 1 0.00
K127Q 1 0.00 K127S 1 0.00 - K127T 3 0.00 K127Y 1 0.00 Y128D 8948
1.42 ++ Y128E 0 0.00 Y128H 10887 1.73 ++ Y128K 452 0.25 Y128Q 11415
1.81 ++ R129D 3093 0.49 + R129E 15209 2.42 + R129K 411 0.23 R129N
573 0.32 + R129S 46 0.03 N130D 18652 2.96 + R134D 1998 0.32 - R134E
21604 3.43 ++ R134K 830 0.47 R134L 9016 5.08 + R134P 125 0.07 R134Q
1 0.00 - R134S 4314 2.43 ++ A135D 5 0.00 - A135E 28974 4.61 ++
A135S 29539 4.70 ++ H139R 27483 4.37 +++
[0186] Preferred modifications include those modifications that
increase the expression yield in 293T cells by at least 2-fold,
including but not limited to L21N, K39A, K39S, Q53D, Q53T, I57L,
E60Q, A63E, A63Q, A63R, A63S, Y65D, Y65N, A72D, A72E, A72H, A72K,
A72S, F73H, N76A, N76D, N76S, N76T, Y78D, Y78N, Y78T, I86A, I86D,
I86E, I86K, I86Q, F93H, E97D, T98K, N110D, A111S, Y116H, F117H,
F117Y, S120D, R129D, N130D, R134E, R134L, R134S, A135E, A135S, and
H139R. Especially preferred modifications include those
modifications that increase the expression yield in 293T cells by
at least 5-fold, including but not limited to A63Q, A63S, Y65D,
Y65N, A72D, F73H, Y78N, Y78T, I86A, and R134L. Furthermore, a
silent mutation at Y116 from codon TCA to codon TAT was observed to
increase the expression yield in 293T cells by 2.6-fold.
[0187] Additional expression protocols were used for different
scales (96-, 48-, 24-, 12-, or 6-well dishes as well as 10 cm or 15
cm plates), serum-free expression, and expression in alternate
hosts (CHO and BRK-21).
[0188] Expression yields of selected Library 1 variants were
determined for 293T, CHO, and BRK21 cells, as shown below.
Expression of the wild type Image clone does vary between
expression hosts. However, the relative expression yields for the
different variants tend to be improved across hosts, indicating
that a variant that improves expression yield in one host tends to
improve expression yield in other hosts. TABLE-US-00013 TABLE 13
Expression yields of selected Library 1 variants in different
expression hosts Variant Variant Variant Variant Yield vs. Image
Variant 23: 154: 249: 117: 80: Image in clone L21G Q53T Y65N Y78H
F93H 293T 1 3.8 1.9 7.2 2.3 8.5 CHO 1 0.9 1.4 6.8 1.1 6.5 BRK21 1
1.0 1.4 10.2 1.3 5.8
[0189] Relative expression yield was also tested as a function of
DNA dose (5.0, 2.5, and 1.0 ug were tested) and was found to be
dose-independent.
Example 10
Characterization of the Receptor Binding Affinity of the BMP-7
Library 1 variants
[0190] The affinity of human and variant BMP-7 for several BMP
receptors (ActRIa, BMPRIb, ActRIIa, and BMPRII) was measured using
an ELISA-like assay, described below. 96-well plates were coated
with a capture antibody (R&D Systems BMP-7 ELISA Duoset DY354,
part # 840971) by diluting the antibody to 2 .mu.g/mL in PBS,
applying 50 .mu.L/well, and incubating overnight or over the
weekend at 4.degree. C. in a humidified chamber. Excess liquid was
removed from each plate. The plates were blocked by adding 175
.mu.L blocking solution (1% BSA and 5% sucrose in PBS) to each well
and incubating 1-2 hours at room temperature in a humidified
chamber. The plates were then washed using an automated plate
washer. 50 .mu.L of BMP-7 containing solution (for example, diluted
conditioned media obtained from the BMP-7 expression protocol
above, or purified recombinant human BMP-7 of a known
concentration) was added to each well and incubated 1.5-2 hours at
room temperature in a humidified chamber. The plates were then
washed using an automated plate washer. 50 .mu.L of 5 .mu.g/mL BMP
receptor-Fc fusion in PBS was added to each well and incubated 1.5
hours at room temperature in a humidified chamber. The plates were
then washed using an automated plate washer. 50 .mu.L of 1:10,000
diluted anti-human IgG-HRP conjugate in secondary antibody dilution
buffer (1% BSA in PBS, filtered through a 0.2 50 .mu.m filter) was
added to each well and incubated 30 minutes at room temperature in
a humidified chamber. The plates were then washed using an
automated plate washer. 50 .mu.L of pre-mixed TMB substrate (BD
Pharmingen # 555214) was added to each well and incubated for 10-20
minutes at room temperature in the dark. 25 .mu.L of 2N
H.sub.2SO.sub.4 was added to each well. Absorbance readings at 450
nm were taken using a 540 nm wavelength correction.
[0191] The table below shows the receptor binding affinity of a
selection of BMP-7 variants relative to the wild type protein
(normalized to 1.0). Note that the assays were performed using a
fixed volume of conditioned media rather than a specific
concentration of protein, so differences in expression levels as
well as differences in receptor binding affinity may affect the
results. Data is shown only for variants with expression yields
greater than 10.0 ng/mL. TABLE-US-00014 TABLE 14 Receptor binding
affinity of BMP-7 Library 1 variants. ELISA Conc. BMPRIa BMPRIb wt
res# variant (ng/mL) ActRIIa binding BMPRII binding binding binding
M 23 S 15.20 2.20 2.41 0.88 1.09 W 52 A 37.78 1.69 1.25 0.92 0.33 W
55 N 10.93 0.41 0.82 0.35 0.00 F 73 S 15.45 2.54 2.84 0.90 0.25 F
73 D 26.89 2.50 3.05 0.33 0.64 F 73 Q 74.76 1.87 2.85 1.05 1.71 F
73 E 138.00 1.87 1.77 0.55 1.23 F 73 A 153.25 1.13 0.82 0.55 1.05 F
73 A 84.87 0.56 0.25 0.00 0.53 Y 78 D 517.04 1.93 2.72 1.05 2.01 Y
78 T 714.26 1.63 2.66 0.87 2.40 Y 78 S 789.68 1.58 1.77 1.29 2.75 L
21 N 414.21 1.03 1.73 1.57 2.81 L 21 G 12.36 1.50 2.30 2.53 3.65 Y
44 N 13.63 0.45 1.46 2.05 1.81 F 73 T 192.64 2.29 3.41 2.04 2.86 Y
44 T 21.46 0.36 0.25 1.09 1.33 W 55 R 47.80 1.20 1.52 1.75 2.17 F
73 G 22.42 1.41 1.55 1.36 1.64 F 73 K 24.61 0.61 0.38 0.69 1.14 F
73 G 172.77 1.73 1.26 1.16 1.53 F 73 T 154.57 1.92 1.93 1.12 1.44 Y
78 R 263.91 2.26 3.13 1.36 2.39 Y 78 P 1249.23 0.92 0.85 0.75 0.69
I 86 K 2214.38 0.97 1.02 0.81 0.69 I 86 E 2555.62 1.00 1.13 0.66
0.69 I 86 Q 3007.62 0.96 1.07 0.66 0.82 L 90 R 338.46 0.47 0.13
0.29 0.32 L 90 T 647.00 0.66 0.29 0.48 0.64 L 90 E 234.92 0.24 0.18
0.20 0.27 L 90 R 356.38 0.47 0.12 0.22 0.48 F 93 A 11.61 0.11 0.09
0.10 0.24 F 93 E 645.77 0.79 0.62 0.83 0.99 F 93 D 52.77 0.80 0.62
0.96 0.91 F 93 T 287.00 0.79 0.81 0.77 0.88 L 115 A 24.24 0.01
-0.01 -0.02 0.17 F 117 R 603.69 0.35 0.72 0.47 0.71 F 117 K 329.23
0.34 0.20 0.43 0.62 L 90 H 61.98 0.47 0.20 0.36 0.55 L 90 P 109.40
0.75 0.17 0.46 0.52 L 90 G 91.95 0.87 0.28 0.55 0.65 F 93 H 233.85
0.98 1.20 1.03 1.04 F 93 G 23.58 0.63 0.35 0.60 0.53 Y 116 Y
1921.77 1.02 1.12 1.07 0.79 F 117 S 42.22 0.11 -0.04 0.15 -0.08 F
117 Y 1682.15 1.03 0.96 1.07 0.73 V 123 G 304.85 0.53 0.22 0.67
0.17 L 21 R 681.69 0.84 0.66 0.83 1.01 L 21 E 332.00 0.25 0.07 0.12
0.16 L 21 D 999.08 0.94 0.94 1.01 1.52 M 23 N 600.08 0.94 0.90 0.98
1.36 V 26 G 604.77 1.07 1.00 1.08 1.66 V 26 N 976.38 1.00 0.97 1.14
1.43 V 26 E 1020.54 1.02 0.73 1.02 1.38 W 52 P 407.15 0.07 0.01
0.03 0.14 W 55 A 26.22 0.09 0.03 0.10 0.23 I 57 I 525.77 1.05 1.20
1.01 1.53 I 57 L 23.04 0.90 0.40 0.92 1.17 F 73 N 321.23 0.89 0.85
0.51 0.95 Y 78 H 1489.38 1.07 1.47 1.36 1.34 I 86 D 1703.85 1.09
1.31 1.03 1.18 I 94 Q 854.46 1.10 1.32 0.74 0.74 Y 128 K 452.40
1.11 1.78 1.40 1.57 K 39 N 69.00 1.08 0.99 1.98 1.63 K 39 A 8898.00
1.06 1.51 3.48 2.86 K 39 S 7148.00 1.04 1.37 2.77 2.23 R 48 E 73.90
0.34 -0.13 0.45 -0.51 R 48 Q 743.20 0.80 0.15 1.06 0.85 R 48 N
410.20 0.93 0.13 1.87 0.43 R 48 H 42.82 1.02 0.58 3.15 3.58 Q 53 G
82.88 0.99 1.37 3.70 3.95 Q 53 R 149.28 0.81 0.96 1.26 1.41 Q 53 E
1236.80 0.98 0.72 2.30 2.78 Q 53 D 6876.00 0.89 1.01 2.20 3.74 Q 53
K 485.40 0.73 0.14 0.70 2.03 Q 53 T 5296.00 1.24 1.28 0.80 0.90 Q
53 T 4072.00 1.14 1.23 0.73 0.85 Q 53 S 5978.00 1.04 1.26 0.66 0.68
E 60 R 122.78 0.68 0.77 0.35 0.23 E 60 P 145.10 0.94 1.01 0.47 0.53
E 60 H 128.78 0.86 1.25 0.34 0.42 E 60 R 148.04 0.80 0.40 0.43 0.50
N 76 T 4576.00 1.11 0.97 0.94 0.98 N 76 D 7534.00 1.13 0.87 0.84
0.80 N 76 Y 813.80 0.92 0.60 0.46 0.46 N 76 N 4998.00 1.05 1.12
0.72 0.78 N 76 A 5570.00 0.96 0.95 0.79 0.81 N 76 S 8816.00 0.99
0.98 0.76 0.85 S 77 A 24.84 0.91 0.80 0.55 0.76 S 77 T 15.45 1.15
0.97 0.77 0.79 S 77 E 45.54 1.17 0.98 0.81 0.94 S 77 P 691.00 1.00
0.91 0.58 0.79 S 77 A 1194.00 1.05 0.84 0.85 0.92 S 77 H 48.80 0.42
0.08 0.29 0.30 K 126 E 24.45 0.26 0.00 0.23 0.22 K 126 Q 145.46
0.81 0.28 0.46 0.57 R 129 N 573.40 1.10 1.40 0.89 1.03 R 129 D
1305.00 1.13 1.86 0.93 1.16 R 129 S 45.52 0.62 0.26 0.32 0.37 R 129
K 410.80 1.12 0.78 0.69 0.77 R 134 K 830.00 1.18 0.91 1.05 1.11 R
134 R 249.28 1.09 0.81 0.87 0.90 R 134 S 4314.00 1.17 1.07 1.10
1.15 K 39 T 19.26 0.98 0.75 0.74 0.86 K 39 D 18.87 0.88 0.72 0.67
0.82 Q 53 A 27.33 0.84 0.26 0.54 0.65 E 60 S 97.16 1.02 0.88 0.74
0.87 S 77 T 1047.80 0.99 0.93 0.94 0.90 S 77 P 1302.20 0.92 0.85
0.68 0.95 K 126 G 55.32 0.53 0.12 0.30 0.35 R 134 P 125.14 0.89
0.50 0.54 0.60 R 134 R 927.00 1.11 1.02 0.86 0.98 R 134 L 9016.00
0.99 1.00 1.03 1.02
[0192] To further characterize receptor binding, dose-response
binding assays, using 12-point serial dilutions from conditioned
media, were conducted for selected Library 1 variants.
[0193] Next, dissociation constants (K.sub.D below) were calculated
for each variant using the nonlinear regression--one site
hyperbolic binding model in Prism. Note that the experiment was
repeated for the wild type protein. The relative binding constants
may be compared to determine whether the specificity of each
variant is appreciably different from wild type. TABLE-US-00015
TABLE 16 Dissociation constants for BMP-7 Library 1 variants
ActRIIa BMPRII BMPRIa BMPRIb variant K.sub.D K.sub.D K.sub.D
K.sub.D WT 0.40 1.28 1.61 1.77 WT 0.28 0.98 1.53 0.33 L21G 0.43
2.01 1.65 0.77 L21K 0.84 3.09 2.92 1.75 L21N 3.38 5.01 7.48 5.02
L21R 0.31 2.14 3.32 1.60 M23G 0.31 0.88 2.89 2.53 M23N 0.06 0.99
1.79 1.07 M23R 0.00 2.16 1.69 1.75 M23S 0.58 3.03 2.77 1.39 V26E
2.39 15.80 9.89 6.13 V26G 1.71 3.93 5.76 12.74 V26K 2.42 18.29 7.02
6.98 V26N 1.88 9.33 7.94 5.45 K39A 0.08 0.68 1.97 1.13 K39S 0.12
0.88 1.65 1.11 Y44A 1.57 4.27 5.54 2.39 Y44D 2.08 44.04 3.71 2.18
Y44G 1.10 21.39 4.98 2.56 Y44N 0.80 3.34 2.92 2.63 Y44P 0.80 11.56
6.46 1.67 Y44S 0.53 6.26 3.48 1.82 R48H 0.23 2.38 2.57 1.04 R48N
0.26 3.14 2.78 1.10 R48Q 1.42 5.37 5.17 5.16 W52A 0.45 8.16 5.00
1.61 Q53A 0.52 2.04 2.92 2.01 Q53D 1.32 5.37 5.22 4.45 Q53G 0.06
0.82 0.73 0.72 Q53H 0.53 7.97 3.38 2.13 Q53K 0.21 1.31 1.57 1.46
Q53S 0.13 1.12 1.36 1.07 Q53T 0.09 0.80 1.36 1.13 W55N 0.55 5.52
5.99 1.99 I57H 0.56 5.34 3.70 2.47 I57I 0.13 1.23 1.36 0.67 I57L
0.09 0.71 1.32 0.81 E60K 0.70 3.71 4.03 3.25 E60Q 0.49 2.32 2.93
2.11 E60R 0.41 1.01 2.76 2.58 A63Q 0.51 0.98 2.20 1.09 A63S 0.61
2.29 2.90 1.30 Y65D 0.45 1.11 2.19 1.11 A72D 0.46 1.56 2.68 1.30
A72H 0.54 2.55 1.98 1.71 A72N 0.81 3.21 4.02 1.25 A72S 0.35 1.50
2.48 1.04 F73E 0.72 34.63 4.25 1.79 F73S 0.18 1.72 2.33 1.08 Y78H
0.10 1.05 1.46 1.12 Y78R 0.94 11.65 3.90 1.68 Y78S 0.19 1.10 1.40
0.98 N83P 0.18 0.77 1.39 1.78 I86A 0.16 1.12 3.55 1.12 I86D 0.85
11.19 24.59 8.03 Q88E 1.33 8.96 9.65 7.32 L90N 1.11 12.59 7.26 5.23
F93D 0.40 3.02 1.18 1.20 F93E 0.73 17.88 3.57 1.33 F93G 0.43 1.21
6.15 5.35 F93H 0.14 0.84 1.35 0.71 F93Q 0.94 4.44 6.25 3.46 F93R
1.11 8.96 9.30 5.95 F93S 0.61 1.89 1.11 1.37 F93T 0.74 1.85 3.94
1.34 I94E 3.02 9.73 14.20 8.08 I94H 0.93 9.03 7.10 4.62 I94K 1.07
1.07 4.74 3.05 I94R 0.68 1.07 3.70 1.78 N95K 2.13 1.49 8.31 1.46
N95R 5.35 14.15 14.18 11.13 E97D 0.38 1.27 3.35 1.40 E97K 0.65 2.36
7.88 2.72 E97R 0.24 1.38 2.22 0.31 T98A 0.20 1.17 2.09 0.27 T98E
0.14 1.41 1.95 0.35 T98K 0.18 1.04 1.72 0.33 T98X 0.15 1.32 1.60
0.34 A105V 0.15 1.01 1.11 0.41 Q108D 0.14 0.84 2.01 0.42 Q108S 0.15
1.25 0.95 0.46 N110D 0.20 0.35 0.62 0.34 N110E 0.28 0.68 0.98 0.39
Y116H 0.28 1.96 2.12 0.41 Y116Y 0.25 1.06 2.20 0.93 F117H 0.16 0.84
1.43 0.28 F117R 0.65 5.54 4.17 1.17 F117Y 1.26 5.15 5.11 6.81 S120D
0.10 1.07 0.89 0.30 R129D 0.26 0.43 2.22 1.40 R129N 2.66 1.78 7.85
8.90 R134E 0.31 1.07 2.43 0.55 R134L 0.29 1.75 12.33 1.23 R134R
0.39 6.16 4.26 2.72 R134S 1.48 3.19 5.21 4.56 A135E 0.08 1.22 0.99
0.38 A135S 0.17 1.31 0.67 0.44 H139R 0.15 1.41 1.00 0.37
[0194] Based on the above-described results, the vast majority of
the variants have receptor binding affinities that are similar to
wild type BMP-7. The following variants appear to have altered
specificity for the type II receptors: M23N, Q53G, Q53H, and I86D.
Q53H and I86D bind to ActRIIa with similar affinity to wild type,
but bind BMPRII with approximately 10-fold reduced affinity
relative to wild type. M23N and Q53G bind to ActRIIa with
approximately 10-fold increased affinity relative to wild type, but
bind BMPRII with similar affinity to wild type.
Example 11
Characterization of Library 1 BMP Variants Using the C2C12
Bioassay
[0195] The biological activity of human and variant BMP-7 molecules
was measured using the C2C12 bioassay. C2C12 cells are a mouse
myoblastic cell line that differentiates in response to BMPs such
as BMP-7. C2C12 cells were trypsinized and diluted to approximately
60,000 cells/mL in C2C12 media (DMEM, 4 mM L-glutamine, 1.5 g/L
sodium bicarbonate, 4.5 g/L glucose, 10% FBS, and antibiotics). 50
.mu.L (3000 cells) were dispensed into each well of a 96-well plate
and incubated overnight at 37.degree. C. The next day, 50 .mu.L of
BMP-7 containing solution (for example, diluted conditioned media
obtained from the BMP-7 expression protocol above, or purified
recombinant human BMP-7 of a known concentration) was added to each
well; each sample was tested in duplicate. The plates were
incubated for 3 days at 37.degree. C. The plates were then washed
twice with 150 .mu.L TBS (50 mM Tris pH 7.5, 150 mM NaCl). 25 .mu.L
TBS with 1% Triton-X100 was added to each well and the plate was
incubated for 10-20 minutes at 4.degree. C. 100-150 .mu.L CSPD
SapphireII luminescent alkaline phosphatase substrate (Applied
Biosystems #T2210) was added to each well and incubated at room
temperature in the dark. Luminescence readings were obtained for
each well using the TopCount plate reader. Luminescence of the
BMP-7 variants were compared to the luminescence of known
quantities of recombinant human BMP-7 in order to determine the
relative biological activity of the variants.
[0196] The table below shows the bioactivity of a selection of
BMP-7 variants relative to the wild type protein (normalized to
1.0). Note that the assays were performed using a fixed volume of
conditioned media rather than a specific concentration of protein,
so differences in expression levels as well as differences in
receptor binding affinity may affect the results. Data is shown
only for variants with expression yields greater than 10.0 ng/mL.
TABLE-US-00016 TABLE 17 Bioactivity of BMP-7 Library 1 variants
var# wt res # var ELISA Conc. (ng/mL) C2C12 Bioactivity 1 M 23 S
15.20 8.65 6 W 52 A 37.78 0.11 9 W 55 N 10.93 0.06 13 F 73 S 15.45
0.11 14 F 73 D 26.89 0.03 15 F 73 Q 74.76 0.20 16 F 73 E 138.00
0.09 17 F 73 A 153.25 0.18 18 F 73 A 84.87 -0.08 19 Y 78 D 517.04
0.13 20 Y 78 T 714.26 0.17 21 Y 78 S 789.68 0.09 22 L 21 N 414.21
1.21 23 L 21 G 12.36 4.34 28 Y 44 N 13.63 0.11 30 F 73 T 192.64
0.16 31 Y 44 T 21.46 0.13 33 W 55 R 47.80 0.51 37 F 73 G 22.42 0.07
39 F 73 K 24.61 0.11 40 F 73 G 172.77 0.13 41 F 73 T 154.57 0.17 42
Y 78 R 263.91 2.50 43 Y 78 P 1249.23 0.00 49 I 86 K 2214.38 0.01 50
I 86 E 2555.62 0.02 51 I 86 Q 3007.62 0.05 52 L 90 R 338.46 0.04 53
L 90 T 647.00 0.01 54 L 90 E 234.92 0.00 55 L 90 R 356.38 0.00 56 F
93 A 11.61 0.05 57 F 93 E 645.77 6.14 58 F 93 D 52.77 6.05 59 F 93
T 287.00 5.61 63 L 115 A 24.24 -0.01 66 F 117 R 603.69 0.06 67 F
117 K 329.23 0.08 76 L 90 H 61.98 0.01 77 L 90 P 109.40 -0.01 78 L
90 G 91.95 -0.01 80 F 93 H 233.85 5.78 82 F 93 G 23.58 5.38 85 Y
116 Y 1921.77 2.34 86 F 117 S 42.22 0.00 87 F 117 Y 1682.15 1.84 88
V 123 G 304.85 0.31 97 L 21 R 681.69 1.20 98 L 21 E 332.00 0.00 99
L 21 D 999.08 0.89 100 M 23 N 600.08 1.54 103 V 26 G 604.77 2.13
104 V 26 N 976.38 2.75 105 V 26 E 1020.54 0.97 106 W 52 P 407.15
0.00 109 W 55 A 26.22 -0.01 111 I 57 I 525.77 1.66 112 I 57 L 23.04
0.05 116 F 73 N 321.23 -0.01 117 Y 78 H 1489.38 2.67 119 I 86 D
1703.85 0.20 120 I 94 Q 854.46 0.00 125 Y 128 K 452.40 0.12 137 K
39 N 69.00 0.13 140 K 39 A 8898.00 1.27 141 K 39 S 7148.00 0.96 143
R 48 E 73.90 0.00 144 R 48 Q 743.20 0.10 145 R 48 N 410.20 0.01 147
R 48 H 42.82 0.33 148 Q 53 G 82.88 1.00 149 Q 53 R 149.28 0.02 150
Q 53 E 1236.80 0.08 151 Q 53 D 6876.00 1.67 152 Q 53 K 485.40 0.04
153 Q 53 T 5296.00 0.72 154 Q 53 T 4072.00 1.20 155 Q 53 S 5978.00
1.52 156 E 60 R 122.78 0.17 159 E 60 P 145.10 0.73 160 E 60 H
128.78 0.32 161 E 60 R 148.04 0.28 162 N 76 T 4576.00 0.12 163 N 76
D 7534.00 0.01 164 N 76 Y 813.80 0.00 165 N 76 N 4998.00 1.18 166 N
76 A 5570.00 0.05 167 N 76 S 8816.00 0.20 168 S 77 A 24.84 0.32 169
S 77 T 15.45 0.00 170 S 77 E 45.54 -0.01 171 S 77 P 691.00 -0.01
172 S 77 A 1194.00 0.60 173 S 77 H 48.80 0.01 174 K 126 E 24.45
0.01 175 K 126 Q 145.46 0.21 182 R 129 N 573.40 1.50 183 R 129 D
1305.00 3.96 184 R 129 S 45.52 0.05 185 R 129 K 410.80 0.01 186 R
134 K 830.00 0.87 187 R 134 R 249.28 0.50 188 R 134 S 4314.00 3.76
190 K 39 T 19.26 0.06 191 K 39 D 18.87 0.01 192 Q 53 A 27.33 0.02
193 E 60 S 97.16 0.44 197 S 77 T 1047.80 0.01 199 S 77 P 1302.20
0.00 200 K 126 G 55.32 0.00 204 R 134 P 125.14 0.17 205 R 134 R
927.00 3.57 206 R 134 L 9016.00 3.76
[0197] Interestingly, a number of the library 1 variants have
significantly greater bioactivity than the wild type Image clone.
This observed increase in activity is likely due to increased
expression yield, although additional factors including but not
limited to altered stability, solubility, or receptor binding
affinity may also influence the observed bioactivity. Substitutions
that increase bioactivity by at least 2-fold relative to wild type
include, but are not limited to, L21G, M23S, V26G, V26N, Y78H,
Y78R, F93D, F93E, F93G, F93H, F93T, R129D, R134L, and R134S.
Example 12
Double Mutant Variants: BMP-7 Library 2
[0198] The point mutations from selected Library 1 variants were
combined to yield a library of double mutants, referred to as
Library 2. Methods for making and screening the Library 2 variants
are as for the Library 1 variants described above. C2C12 bioassay
data was determined at a single point by diluting conditioned media
1:66; due to the low expression yield of the Image clone, its
bioassay signal is at background at this dilution. TABLE-US-00017
TABLE 18 Expression yield and bioassay data, BMP-7 Library 2
variants. Western Blot 293T Fold CHO Fold Band ELISA change: ELISA
change: C2C12 Inten- Variant Name (ng/mL) 293T (ng/mL) CHO @ 1:66
sity L21G-Y65N 4261.8 2.1 73.7 3.6 0.09 ++ L21G-F93H 23923.5 12.0
20.8 1.1 0.23 ++ L21R-Y65N 4371.2 2.2 59.9 2.9 0.16 ++ M23R-Y65N
5468.8 2.7 182.3 8.8 0.20 ++ K39A-F93H 37258.8 18.6 222.5 10.8 0.66
++ K39S-Y65N 5259.4 2.6 39.4 1.9 0.13 ++ K39S-A72D 3703.5 1.9 0.7
0.0 0.05 ++ K39S-Y78H 18617.6 9.3 173.8 8.4 0.02 ++ K39S-I86A
4231.2 2.1 16.3 0.8 0.05 ++ K39S-I94R 4.9 0.0 0.3 0.0 0.04 ++
K39S-F93S 3256.5 1.6 45.7 2.2 1.23 +++ K39S-Q108D 5472.9 2.7 42.4
2.1 0.07 +++ K39S-N110D 19964.7 10.0 171.8 8.3 0.49 ++ K39S-S120D
21147.1 10.6 134.3 6.5 0.46 +++ K39S-R129D 2048.8 1.0 34.0 1.6 0.28
++ K39S-N130D 3918.2 2.0 284.5 13.8 0.20 ++ K39S-R134E 20564.7 10.3
6.2 0.3 0.28 ++ K39S-R134S 18847.1 9.4 35.1 1.7 0.20 ++ K39S-A135E
17694.1 8.8 159.9 7.8 0.03 ++ K39S-A135S 2783.5 1.4 22.8 1.1 0.03
++ K39S-H139R 2868.8 1.4 37.2 1.8 0.03 ++ Q53D-Y65N 22552.9 11.3
88.7 4.3 0.02 ++ I57L-Y65N 18982.4 9.5 158.7 7.7 0.02 ++ Y65N-Y78H
27923.5 14.0 197.3 9.6 0.13 ++ Y65N-Y78R 20076.5 10.0 185.7 9.0
0.06 ++ Y65N-S120D 23794.1 11.9 218.3 10.6 0.66 +++ Y65N-A135S
22023.5 11.0 51.5 2.5 0.12 ++ A72D-F93H 23329.4 11.7 369.2 17.9
0.17 ++ Y78H-F93H 18117.6 9.1 395.7 20.5 0.36 ++ Y78H-Q108D 35176.5
17.6 0.7 0.0 0.27 ++ Y78H-Y116H 23688.2 11.8 70.8 3.2 0.01 +
Y78H-F117Y 21794.1 10.9 62.3 5.5 0.07 ++ Y78H-S120D 27864.7 13.9
179.6 12.5 0.34 ++ Y78H-R134E 40129.4 20.1 410.1 21.6 0.72 ++
Y78H-R134S 49617.6 24.8 36.3 2.4 0.68 ++ Y78H-A135E 49729.4 24.9
167.6 9.6 0.03 ++ Y78H-A135S 34458.8 17.2 43.4 2.2 0.05 ++
Y78H-H139R 37770.6 18.9 40.5 2.1 0.14 ++ F93H-F117Y 24747.1 12.4
296.0 14.4 0.40 ++ F93H-S120D 35758.8 17.9 276.1 13.4 1.14 ++
F93H-R134S 33800.0 16.9 378.7 18.4 1.21 ++ F93H-H139R 39929.4 20.0
279.5 13.6 0.53 ++
[0199] As may be seen above, a number of the Library 2 variants
have significantly increased expression yield, in both 293T and CHO
cells, relative to the wild type Image clone. Preferred variants
show at least a 10-fold increase in at least one expression host;
examples of such variants include but are not limited to L21G/F93H,
K39A/F93H, K39S/N110D, K39S/S120D, K39S/N130D, K39S/R134E,
Q53D/Y65N, Y65N/Y78H, Y65N/Y78R, Y65N/S120D, Y65N/A135S, A72D/F93H,
Y78H/F93H, Y78H/Q108D, Y78H/Y116H, Y78H/F117Y, Y78H/S120D,
Y78H/R134E, Y78H/R134S, Y78H/A135E, Y78H/A135S, Y78H/H139R,
F93H/F117Y, F93H/S120D, F93H/R134S, F93H/H139R. Especially
preferred variants show at least a 10-fold increase in expression
yield in both 293T and CHO cells; examples of such variants include
but are not limited to K39A/F93H, Y65N/S120D, A72D/F93H,
Y78H/S120D, Y78H/R134E, Y78H/A135E, F93H/F117Y, F93H/S120D,
F93H/R134S, and F93H/H139R.
Example 13
Expression Yield of Triple, Quadruple, and Higher-Order Mutants
[0200] Triple, quadruple, and higher order mutants of BMP-7 were
made and tested as described above. A number of these variants
exhibit significantly increased expression yield or significantly
increased bioactivity. Note that ELISA substantially underestimates
the protein concentration of a substantial fraction of these
variants due to decreased antibody binding affinity. TABLE-US-00018
TABLE 19 Expression yield in 293T cells and bioactivity data for
selected triple variants with high expression yield in 293T cells
Fold Fold ELISA Increase Increase Western Conc. Expression C2C12
Blot Band Variant Name (ng/mL) Yield Bioactivity Intensity
K39S/S120D/Y78H 50100.0 96.8 7.1 +++ Y78H/F93H/F117H 43490.0 84.0
23.0 +++ Y78H/F93H/Q108D 42880.0 82.8 21.6 +++ Y78H/F93H/A72D
42590.0 82.3 14.6 +++ K39S/S120D/Q108D 41170.0 79.5 15.6 +++
Y78H/F93H/Y65N 34830.0 79.4 21.3 +++ Y78H/F93H/S120D 40260.0 77.8
26.6 +++ Y78H/R134E/Y65N 40210.0 77.7 42.2 ++++ K39S/S120D/Y65N
34260.0 66.2 17.8 +++ K39S/S120D/A72D 34070.0 65.8 25.9 +++
K39S/S120D/R134E 32320.0 62.4 37.9 +++ K39S/S120D/M23R 32060.0 61.9
14.3 +++ K39S/S120D/H139R 29930.0 57.8 21.2 ++ K39S/S120D/R129D
5910.0 11.4 22.7 +++ Y78H/R134E/M23R 4775.0 9.2 27.7 ++
Y78H/R134E/L21G 4763.0 9.2 28.4 ++ Y78H/R134E/A72D 3269.0 6.3 43.6
++++ K39S/S120D/F93H 2950.0 5.7 2.1 + K39S/S120D/F93S 2031.0 4.6
45.3 +++
[0201] All of the above triples have expression yields that are at
least 50-fold higher than wild type, and at least 2-fold higher
than the best doubles. Furthermore, the majority of the above
triples have significantly increased bioactivity relative to wild
type and the best doubles. Triple variants with especially high
bioactivity include, but are not limited to, K39S/F93S/S120D,
K39S/S120D/R134E, Y65N/Y78H/R134E, and A72D/Y78H/R134E.
TABLE-US-00019 TABLE 20 Expression yield in CHO-K1 cells and
bioactivity data for selected triple variants with high expression
yield in CHO-K1 cells Fold Fold ELISA Increase Increase Western
Conc. Expression C2C12 Blot Band Variant Name (ng/mL) Yield
Bioactivity Intensity Y78H/R134E/Y65N 316.2 17.1 3.6 -
Y78H/F93H/S120D 316.1 17.1 2.8 - K39S/S120D/R134E 245.7 13.3 21.8 +
Y78H/R134E/A72D 232.1 12.5 14.7 + K39S/S120D/Q108D 219.8 11.9 2.4 -
Y78H/F93H/Y65N 200.0 10.8 1.6 - Y78H/R134E/L21G 198.0 10.7 2.8 -
Y78H/F93H/Q108D 115.3 6.2 2.9 - Y78H/F93H/A72D 107.1 5.8 2.0 -
Y78H/R134E/M23R 78.8 4.3 6.4 - Y78H/F93H/F117H 58.7 3.2 1.0 -
K39S/S120D/A72D 46.8 2.5 1.6 - K39S/S120D/R129D 42.9 2.3 28.0 +
K39S/S120D/F93S 38.4 2.1 78.7 + K39S/S120D/H139R 33.8 1.8 1.5 -
[0202] Preferred variants with dramatic increases in CHO-K1
expression yield or C2C12 bioactivity include but are not limited
to K39S/F93S/S120D, K39S/S120D/R129D, K39S/S120D/R134E,
Y65N/Y78H/R134E, and Y78H/F93H/S120D.
[0203] Additional triple mutants were generated to determine the
impact of different substitutions on receptor and inhibitor binding
specificity. These variants included the Y65N and S120D
substitutions, which confer increased expression yield and do not
significantly affect receptor or inhibitor binding, and one
additional substitution that may alter binding specificity. Triples
comprising Y65N/S120D, and one of the following substitutions were
made: M23R, R48H, R48N, R48Q, Q53G, Q53H, Q53K, Q53T, E60R, F73S,
F73T, Y78D, Y78S, Y78T, 186D, K126R, Y128D, Y128H, and Y128Q.
Example 14
Purification of BMP-7 Variants
[0204] Y65N/S120D was partially purified using conventional
chromatography. Heparin-sulphate sepharose (17-0407-01) was
equilibrated in PBS. Conditioned media containing the Y65N/S120D
variant was diluted 1:1 with 40 mM phosphate pH 6.5 filtered
through a 0.45 micron filter, loaded onto the column, washed with
2-3 column volumes of PBS, and eluted in a single isocratic step
with PBS/1M NaCl. The heparin bound fractions were dialyzed into 20
mM phosphate, 50 mM NaCl pH 7.0, loaded onto a SP-sepharose column,
and eluted with a linear gradient (0-100% PBS/1M NaCl). A second
purification protocol was used for larger scale purification of
variants 457 (K39S/F93S), 471 (K39S/N130D), 492 (Y65N/Y78H), 504
(Y65N/S120D), 526 (K39S/S120D/R134E), and 565 (Y65N/F93T/R129D).
Conditioned media was diluted 1:1 at neutral pH to lower the salt
concentration to .about.75 mM and loaded onto a SP-sepharose
column. The column was then washed in 75 mM salt and then 300 mM
salt, and BMP-7 was eluted with 1M salt.
Example 15
Receptor, Antibody, and Inhibitor Binding of Selected Variants
[0205] The binding of selected variants to BMP receptors,
antibodies, and inhibitors was characterized using a fluorescence
binding assay and the AlphaScreen.TM. assay. BMP-7 variant
Y65N/S120D, partially purified as described above, was labeled with
the dye AlexaFluor 568 (Molecular Probes). Small-scale (25 uL)
reactions were performed using 15 uM BMP-7 and dye concentrations
ranging from 0.3 uM to 1000 uM. Reactions using 333 uM and 10 uM
dye then were performed using 750 uL protein. The reaction was
quenched with Tris pH 8.0 and cleaned up using a PD-10 desalt
column; the second fraction was used in the experiments described
below. BMP-7 was also labeled with C6-FXS, a FITC-derived
fluorophore with a 6-carbon spacer between the fluor and the NHS
group. Labeling conditions with various ratios of protein to dye
were tested, in 20 mM PO4, 500 mM NaCl, pH 7 solution. After
establishing labeling conditions, 500 ug Y65N/S120D was labeled.
Excess dye was removed by centrifugation and a PD-10 desalt
column.
[0206] The flourescently labeled BMP-7 was added to serial
dilutions of receptor/Fc fusions of the BMP-7 receptors ActRIa,
BMPRIa, BMPRIb, ActRIIa, ActRIIb, and BMPRII (R&D Systems).
Experiments were also performed in which the fluorescently labeled
BMP-7 was added to serial dilutions of noggin/Fc or gremlin
(R&D Systems). The labeled BMP-7 and receptor or inhibitor was
allowed to incubate, and the fluorescence polarization and
intensity was measured using a TopCount plate reader. Significant
changes in intensity or anisotropy were observed for all of the
receptors and inhibitors tested, for at least one of the labeled
BMP-7 molecules.
[0207] Binding affinity of different BMP variants to these
receptors and inhibitors may be determined by performing
competition experiments. To perform these experiments, labeled
BMP-7 and receptor or inhibitor are combined in amounts that yield
an appreciable change in anisotropy or intensity relative to free
labeled BMP-7. Then, varying amounts of a second, unlabeled BMP-7
molecule are added and the change in anisotropy or polarization is
measured. The EC50 is then given by the concentration of competitor
when half of the labeled BMP-7 is bound and half is free.
[0208] The Y65N/S120D variant of BMP-7 was also biotinylated for
use in AlphaScreen assays using NHS-biotin. Bioactivity of the
biotinylated protein was confirmed. AlphaScreen assays were
performed to determine the binding affinity of selected BMP-7
variants for Fc fusions of the receptors BMPRIa, BMPRIb, ActRIIa,
and BMPRII and the inhibitor noggin (R&D Systems). AlphaScreen
assays were also performed to determine the affinity of selected
variants for an anti-BMP-7 monoclonal antibody (R&D Systems mAb
3541). In all cases, 12-point binding curves were obtained in
triplicate. Each data point corresponds to the luminescence
produced from a solution comprising 10 uL of serially diluted BMP-7
variant, 10 uL receptor, inhibitor, or antibody, 10 uL biotinylated
BMP-7, 10 uL AlphaScreen.TM. acceptor beads, and 10 uL
AlphaScreen.TM. donor beads. Prism was used to calculate EC50
values for selected experiments. TABLE-US-00020 TABLE 21 EC50 of
wild type human BMP-7 (R&D Systems) and BMP-7 variants 504,
526, and 565 for the BMP receptors BMPRIb, ActRIIa, and BMPRII and
the BMP inhibitor noggin, as determined using AlphaScreen .TM.
assays. std. error BMP-7 Receptor or EC(50) EC50 log(EC50)
log(EC50) (logEC50) Fold change variant inhibitor (ug/mL) (ng/mL)
(ug/mL) (ng/mL) ug/mL vs. wt wt BMPRIb 0.0499 49.9 -1.30 1.70 0.136
wt ActRIIa 0.107 107 -0.971 2.03 0.218 wt BMPRII 0.230 230 -0.639
2.36 0.259 wt noggin 2.85 2850 0.455 3.46 0.894 v504 BMPRIb 0.0185
18.5 -1.73 1.27 0.190 0.371 v504 ActRIIa 0.0475 47.5 -1.32 1.68
0.180 0.445 v504 BMPRII 0.222 222 -0.653 2.35 0.567 0.969 v504
noggin 3.40 3400 0.532 3.53 2.32 1.19 v526 BMPRIb 0.0363 36.3 -1.44
1.56 0.172 0.727 v526 ActRIIa 0.0445 44.5 -1.35 1.65 0.148 0.416
v526 BMPRII 0.139 139.4 -0.856 2.14 0.394 0.607 v526 noggin 16.7
16700 1.22 4.22 11.5 5.83 v565 BMPRIb 0.0224 22.4 -1.65 1.35 0.121
0.448 v565 ActRIIa 0.0424 42.4 -1.37 1.63 0.272 0.397 v565 BMPRII
0.409 409 -0.388 2.61 1.02 1.78 v565 noggin 0.392 392 -0.407 2.59
0.471 0.137
[0209] Overall, these three variants have binding affinities that
are similar to wild type. Potentially significant differences
include, but are not limited to, decreased noggin affinity of v526
and increased noggin affinity of v565.
Example 16
Concentration Determination
[0210] Some of the BMP-7 variants, especially those variants with
two or more mutations, exhibit reduced antibody binding affinity.
As a result, ELISA concentration determination systematically
underestimates the concentration of these variants. In order to
obtain more accurate concentrations for these variants, as well as
correction factors for the concentrations determined using ELISA,
multiple concentration determination measurements were performed.
Following purification, the concentration of variants 457, 471,
492, 504, 526, and 565 was assessed using the BCA assay,
densitometry analysis of Coomasie blue stained mature domain
following SDS-PAGE, and Western blotting using a polyclonal
antibody. Wild type BMP-7 (R&D Systems) was used as a
standard.
Example 17
Specific Activity Determination
[0211] The specific activity of five especially preferred BMP-7
variants was determined and compared with the specific activity of
recombinant human BMP-7 purchased from R&D Systems. Equal
concentrations of each protein, as determined above, were tested in
the C2C12 bioassay three times. TABLE-US-00021 TABLE 22 Specific
activity of selected BMP-7 variants. EC50: EC50: EC50: Avg Exp Exp
Exp EC50 Std. Name #1 #2 #3 (ug/mL) dev. Wild type (R&D 2.99
3.53 2.06 2.86 0.74 Systems) 565-Y65N/F93T/R129D 0.1 0.3 0.09 0.16
0.12 526-K39S/S120D/R134E 0.58 1.69 0.49 0.92 0.67 504-Y65N/S120D
2.91 nd 4.85 3.88 1.37 492-Y65N/Y87H 5.91 nd 6.18 6.04 0.19
471-K39S/N130D 1.38 nd 3.99 2.69 1.85 457-K39S/F93S 0.37 1.1 nd
0.74 0.52
[0212] Variants with the F93S and F93T substitutions were found to
have increased specific activity relative to the wild type
protein.
Example 18
Specific Variant Designs
[0213] Quadruple mutant containing variants were designed to
improve the proteins expression yields and ensure the highest
biological activities. The mutant substitutions chosen for these
variants comprise a subset of total variants that either singly, or
in combination improve the properties of BMP-7. The stability and
yield variants were chosen a subset of mutants that show beneficial
protein properties and are located at amino acid residues that do
not make receptor contacts. Furin optimization is defined as a set
of mutant variants, built in either the native or Y65N/S120D
background that have an engineered consensus site for the furin
protease required for normal processing and secretion of BMP-7.
Glycosylation removal variants are mutant BMP-7 proteins, built in
either the native or F93H/R134S background that contain mutations
in the consensus glycosylation site, these variants are predicted
to be aglycosylated. The following table summaries specific variant
BMP-7 proteins created to have the listed properties:
TABLE-US-00022 TABLE 23 Variant Property K39S_S120D_Q108D_F93S High
activity and yield K39S_S120D_R129D_F93S High activity and yield
K39S_S120D_Y65N_F93S High activity and yield K39S_S120D_A72D_F93S
High activity and yield Y78H_R134E_Y65N_F93S High activity and
yield Y78H_R134E_A72D_F93S High activity and yield
K39S_S120D_Q108D_Q108D High activity and yield
K39S_S120D_R129D_Q108D High activity and yield
K39S_S120D_Y78H_Q108D High activity and yield
K39S_S120D_R134E_Q108D High activity and yield
K39S_S120D_A72D_Q108D High activity and yield Y78H_R134E_A72D_Q108D
High activity and yield Y65N_R129D_M23R_Q108D High activity and
yield K39S_S120D_Y65N_R129D High activity and yield
K39S_S120D_Y78H_R129D High activity and yield K39S_S120D_A72D_R129D
High activity and yield Y65N_R129D_M23R_S120D High activity and
yield Y65N_R129D_Q108D_S120D High activity and yield
K39S_S120D_Q108D_Y65N High activity and yield K39S_S120D_R129D_Y65N
High activity and yield K39S_S120D_R134E_Y78H High activity and
yield K39S_S120D_Q108D_R134E High activity and yield
K39S_S120D_Y65N_R134E High activity and yield K39S_S120D_Y78H_R134E
High activity and yield K39S_S120D_A72D_R134E High activity and
yield K39S_S120D_Q108D_M23R High activity and yield
K39S_S120D_R129D_M23R High activity and yield K39S_S120D_Y78H_M23R
High activity and yield K39S_S120D_R134E_M23R High activity and
yield K39S_S120D_A72D_M23R High activity and yield
Y78H_R134E_Y65N_M23R High activity and yield Y78H_R134E_A72D_M23R
High activity and yield Y65N_R129D_Q108D_M23R High activity and
yield Q108D_A72D Stability and yield S120D_A72D Stability and yield
R129D_A72D Stability and yield A135E_A72D Stability and yield
A72D_Q108D Stability and yield S120D_Q108D Stability and yield
Q108D_S120D Stability and yield A135E_S120D Stability and yield
A72D_R129D Stability and yield Q108D_R129D Stability and yield
S120D_R129D Stability and yield Q108D_A135E Stability and yield
S120D_A135E Stability and yield A72D_F93S Stability and yield
A72D_A105V Stability and yield A72D_N110D Stability and yield
A72D_A135S Stability and yield A72D_H139R Stability and yield
F93S_A105V Stability and yield F93S_Q108D Stability and yield
F93S_N110D Stability and yield F93S_S120D Stability and yield
F93S_R129D Stability and yield F93S_R134E Stability and yield
F93S_A135S Stability and yield F93S_H139R Stability and yield
A105V_S120D Stability and yield A105V_R129D Stability and yield
A105V_R134E Stability and yield A105V_A135S Stability and yield
A105V_H139R Stability and yield N110D_S120D Stability and yield
N110D_R129D Stability and yield N110D_R134E Stability and yield
N110D_A135S Stability and yield N110D_H139R Stability and yield
Q108D_R134E Stability and yield Q108D_A135S Stability and yield
Q108D_H139R Stability and yield F117Y_R129D Stability and yield
F117Y_R134E Stability and yield S120D_R134E Stability and yield
S120D_A135S Stability and yield S120D_H139R Stability and yield
L21G_E42D Stability and yield L21G_T98K Stability and yield
L21G_A105V Stability and yield L21G_S120D Stability and yield
L21G_A135S Stability and yield L21G_A135E Stability and yield
L21G_H139R Stability and yield M23R_E42D Stability and yield
M23R_T98K Stability and yield M23R_A105V Stability and yield
M23R_S120D Stability and yield M23R_A135S Stability and yield
M23R_A135E Stability and yield M23R_H139R Stability and yield
E42D_T98K Stability and yield E42D_A105V Stability and yield
E42D_S120D Stability and yield E42D_A135S Stability and yield
E42D_A135E Stability and yield E42D_H139R Stability and yield
T98K_A105V Stability and yield T98K_S120D Stability and yield
T98K_A135S Stability and yield T98K_A135E Stability and yield
T98K_H139R Stability and yield A105V_S120D Stability and yield
A105V_A135S Stability and yield A105V_A135E Stability and yield
A105V_H139R Stability and yield S120D_A135S Stability and yield
S120D_A135E Stability and yield S120D_H139R Stability and yield
WT_P1_QVKKRSKR Furin optimization WT_P2_QVKKRSRR Furin optimization
WT_P3_QVRKRSKR Furin optimization WT_P4_QVRKRSRR Furin optimization
WT_P5_KVKKRSKR Funn optimization WT_P6_KVKKRSRR Furin optimization
WT_P7_KVRKRSKR Furin optimization WT_P8_KVRKRSRR Furin optimization
WT_P9_EVKLRSKR Furin optimization WT_P10_EVKLRSRR Furin
optimization WT_P11_EVRLRSKR Furin optimization WT_P12_EVRLRSRR
Furin optimization Y65N_S120D_QVKKRSKR Furin optimization
Y65N_S120D_QVKKRSRR Furin optimization Y65N_S120D_QVRKRSKR Furin
optimization Y65N_S120D_QVRKRSRR Furin optimization
Y65N_S120D_KVKKRSKR Furin optimization Y65N_S120D_KVKKRSRR Furin
optimization Y65N_S120D_KVRKRSKR Furin optimization
Y65N_S120D_KVRKRSRR Furin optimization Y65N_S120D_EVKLRSKR Furin
optimization Y65N_S120D_EVKLRSRR Furin optimization
Y65N_S120D_EVRLRSKR Furin optimization Y65N_S120D_EVRLRSRR Furin
optimization N80A Glycosylation removal N80C Glycosylation removal
N80D Glycosylation removal N80E Glycosylation removal N80F
Glycosylation removal N80G Glycosylation removal N80H Glycosylation
removal N80I Glycosylation removal N80K Glycosylation removal N80L
Glycosylation removal N80M Glycosylation removal N80P Glycosylation
removal N80Q Glycosylation removal N80R Glycosylation removal N80S
Glycosylation removal N80T Glycosylation removal N80V Glycosylation
removal N80W Glycosylation removal N80Y Glycosylation removal A81P
Glycosylation removal T82A Glycosylation removal T82D Glycosylation
removal T82E Glycosylation removal T82F Glycosylation removal T82G
Glycosylation removal T82H Glycosylation removal T82I Glycosylation
removal T82K Glycosylation removal T82L Glycosylation removal T82M
Glycosylation removal T82N Glycosylation removal T82P Glycosylation
removal T82Q Glycosylation removal T82R Glycosylation removal T82V
Glycosylation removal T82W Glycosylation removal T82Y Glycosylation
removal F93H/R134S/N80A Glycosylation removal F93H/R134S/N80C
Glycosylation removal F93H/R134S/N80D Glycosylation removal
F93H/R134S/N80E Glycosylation removal F93H/R134S/N80F Glycosylation
removal F93H/R134S/N80G Glycosylation removal F93H/R134S/N80H
Glycosylation removal F93H/R134S/N80I Glycosylation removal
F93H/R134S/N80K Glycosylation removal F93H/R134S/N80L Glycosylation
removal F93H/R134S/N80M Glycosylation removal F93H/R134S/N80P
Glycosylation removal F93H/R134S/N80Q Glycosylation removal
F93H/R134S/N80R Glycosylation removal F93H/R134S/N80S Glycosylation
removal F93H/R134S/N80T Glycosylation removal F93H/R134S/N80V
Glycosylation removal F93H/R134S/N80W Glycosylation removal
F93H/R134S/N80Y Glycosylation removal F93H/R134S/A81P Glycosylation
removal F93H/R134S/T82A Glycosylation removal F93H/R134S/T82D
Glycosylation removal F93H/R134S/T82E Glycosylation removal
F93H/R134S/T82F Glycosylation removal F93H/R134S/T82G Glycosylation
removal F93H/R134S/T82H Glycosylation removal F93H/R134S/T82I
Glycosylation removal F93H/R134S/T82K Glycosylation removal
F93H/R134S/T82L Glycosylation removal F93H/R134S/T82M Glycosylation
removal F93H/R134S/T82N Glycosylation removal F93H/R134S/T82P
Glycosylation removal F93H/R134S/T82Q Glycosylation removal
F93H/R134S/T82R Glycosylation removal F93H/R134S/T82V Glycosylation
removal F93H/R134S/T82W Glycosylation removal F93H/R134S/T82Y
Glycosylation removal
Example 18
Variant Designs
[0214] In addition to the preferred embodiments disclosed in Table
23 above, the following variants are also preferred embodiments of
the present invention: TABLE-US-00023 TABLE 24 L21G_V26G L21G_V26N
L21G_M23G L21N_M23G M23G_V26G M23G_V26N 21N_23G_26G A105V A111D
A111S A135D A135E A135S A37E A63E A63Q A63R A63S A72D A72E A72H
A72K A72N A72R A72S A105V D119E D119N D119S D119T D49S D54K D54N
D54S E42D E42Q E42R E42T E60H E60K E60N E60P E60Q E60R E60R E60R
E60S E60T E70A E70Q E97D E97K E97R F117A F117D F117E F117H F117K
F117Q F117R F117S F117Y F73A F73A F73D F73E F73G F73G F73H F73K
F73N F73Q F73R F73S F73T F73T F93A F93D F93E F93G F93H F93H_A105V
F93H_A135E F93H_A72D F93H_F117Y F93H_H139R F93H_I57L F93H_I94R
F93H_K127E F93H_K39A F93H_K39S F93H_L21G F93H_L21R F93H_N130D
F93H_Q108D F93H_R129D F93H_R134E F93H_R134S F93H_R48N F93H_S120D
F93H_Y128D F93H_Y78R F93K F93P F93Q F93R F93S F93T H139R I124A
I124D I124E I124K I124N I124Q I124R I124S I124T I124V I57A I57D
I57E I57H I57I I57K I57L I57N I57P I57Q I57T I57V I86A I86D I86D
I86E I86K I86P I86Q I86T I94A I94E I94H I94K I94K I94P I94Q I94R
I94T K126D K126E K126G K126Q K126R K127A K127D K127E K127E K127H
K127N K127P K127Q K127S K127T K127Y K39A K39A K39D K39E K39G K39N
K39R K39S K39S_A135E K39S_A135S K39S_A72D K39S_F117H K39S_F117Y
K39S_F93S K39S_H139R K39S_I57L K39S_I86A K39S_I94R K39S_L21G
K39S_L21R K39S_M23R K39S_N110D K39S_N130D K39S_Q108D K39S_Q53G
K39S_Q53T K39S_R129D K39S_R134E K39S_R134S K39S_R48N K39S_S120D
K39S_S120D_A72D K39S_S120D_F93H K39S_S120D_F93S K39S_S120D_H139R
K39S_S120D_L21G K39S_S120D_M23R K39S_S120D_Q108D K39S_S120D_R129D
K39S_S120D_R134E K39S_S120D_Y65N K39S_S120D_Y78H K39S_Y116H
K39S_Y128D K39S_Y65N K39S_Y78R K39T L115A L115E L115K L115Q L115T
L125A L125E L125K L125P L125Q L125T L125Y L21D L21E L21G L21G L21H
L21K L21N L21N_M23G_V26N L21R L21S L90A L90E L90G L90H L90K L90L
L90N L90P L90P L90Q L90R L90R
L90R L90S L90T M23D M23G M23K M23N M23R M23S N110D N110E N110H
N122E N122Q N122R N130D N76A N76D N76N N76S N76T N76Y N83P N95D
N95K N95Q N95R Q108D Q108K Q108S Q36E Q36N Q53A Q53D Q53E Q53G Q53H
Q53K Q53R Q53S Q53T Q53T Q88E R129D R129E R129K R129N R129S R134D
R134E R134K R134L R134P R134P R134Q R134R R134R R134S R48D R48E
R48H R48N R48Q S113D S113E S120D S120E S120N S120R S121D S121E
S121K S121N S121T S77A S77A S77A S77D S77E S77H S77K S77N S77P S77P
S77Q S77T S77T T107D T107E T98A T98Del T98E T98E T98K V123A V123A
V123D V123G V123G V123N V123N V123R V123T V123V V26D V26E V26G V26K
V26K V26N V26S V26V W52A W52E W52K W52P W52Q W52T W55A W55A W55E
W55H W55K W55N W55P W55Q W55R W55T Y116A Y116D Y116E Y116H Y116K
Y116Q Y116S Y116T Y116Y Y128D Y128E Y128H Y128K Y128Q Y44A Y44D
Y44E Y44G Y44H Y44K Y44N Y44P Y44Q Y44R Y44S Y44T Y65D Y65E Y65N
Y65N_A105V Y65N_A135E Y65N_A135S Y65N_F117Y Y65N_F93H Y65N_F93T
Y65N_H139R Y65N_I57L Y65N_I94R Y65N_K127E Y65N_K39A Y65N_K39S
Y65N_L21G Y65N_L21R Y65N_M23N Y65N_M23R Y65N_Q108D Y65N_Q53D
Y65N_Q53G Y65N_Q53S Y65N_Q53T Y65N_R129D Y65N_R129D_F117H
Y65N_R129D_F117Y Y65N_R129D_F93H Y65N_R129D_F93S Y65N_R129D_F93T
Y65N_R129D_K39A Y65N_R129D_K39S Y65N_R129D_L21G Y65N_R129D_M23R
Y65N_R129D_Q108D Y65N_R129D_S120D Y65N_R129D_Y78H Y65N_R134E
Y65N_R134S Y65N_S120D Y65N_Y128D Y65N_Y78H Y65N_Y78R Y78A Y78D Y78G
Y78H Y78H_A105V Y78H_A135E Y78H_A135S Y78H_A63S Y78H_A72D
Y78H_F117H Y78H_F117Y Y78H_F93H Y78H_F93H_A72D Y78H_F93H_F117H
Y78H_F93H_F117Y Y78H_F93H_H139R Y78H_F93H_K39A Y78H_F93H_K39S
Y78H_F93H_L21G Y78H_F93H_M23R Y78H_F93H_Q108D Y78H_F93H_R129D
Y78H_F93H_R134S Y78H_F93H_S120D Y78H_F93H_Y65N Y78H_F93T Y78H_H139R
Y78H_I57L Y78H_I94R Y78H_K127E Y78H_K39S Y78H_L21R Y78H_N110D
Y78H_N130D Y78H_Q108D Y78H_Q53G Y78H_Q53S Y78H_R129D Y78H_R134E
Y78H_R134E_A72D Y78H_R134E_F117Y Y78H_R134E_F93H Y78H_R134E_F93S
Y78H_R134E_F93T Y78H_R134E_K39A Y78H_R134E_K39S Y78H_R134E_L21G
Y78H_R134E_M23R Y78H_R134E_Q108D Y78H_R134E_S120D Y78H_R134E_Y65N
Y78H_R134S Y78H_R48N Y78H_S120D
Y78H_Y116H Y78H_Y128D Y78N Y78P Y78R Y78S Y78T Y78Y
[0215] While the foregoing invention has been described above, it
will be clear to one skilled in the art that various changes and
additional embodiments made be made without departing from the
scope of the invention. All publications, patents, patent
applications (provisional, utility and PCT) or other documents
cited herein are incorporated by references in their entirety.
Sequence CWU 1
1
87 1 114 PRT Homo sapiens 1 Gln 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 2 116 PRT Homo sapiens 2 Ser 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 3 138
PRT Homo sapiens 3 Ala 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 4
139 PRT Homo sapiens 4 Ser Ala Ser Ser Arg Arg Arg Gln Gln Ser Arg
Asn Arg Ser Thr Gln 1 5 10 15 Ser Gln Asp Val Ala Arg Val Ser Ser
Ala Ser Asp Tyr Asn Ser Ser 20 25 30 Glu Leu Lys Thr Ala Cys Arg
Lys His Glu Leu Tyr Val Ser Phe Gln 35 40 45 Asp Leu Gly Trp Gln
Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala 50 55 60 Asn Tyr Cys
Asp Gly Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn 65 70 75 80 Ala
Thr Asn His Ala Ile Val Gln Thr Leu Val His Leu Met Asn Pro 85 90
95 Glu Tyr Val Pro Lys Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile
100 105 110 Ser Val Leu Tyr Phe Asp Asp Asn Ser Asn Val Ile Leu Lys
Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130
135 5 139 PRT Homo sapiens 5 Ser Thr Gly Gly Lys Gln Arg Ser Gln
Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met
Ala Ser 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 6 139 PRT Homo sapiens 6 Ala 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 Lys Pro 85 90 95 Asn 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 7 76 PRT Homo sapiens 7 Leu Tyr Met Cys Val
Cys Glu Gly Leu Ser Cys Gly Asn Glu Asp His 1 5 10 15 Cys Glu Gly
Gln Gln Cys Phe Ser Ser Leu Ser Ile Asn Asp Gly Phe 20 25 30 His
Val Tyr Gln Lys Gly Cys Phe Gln Val Tyr Glu Gln Gly Lys Met 35 40
45 Thr Cys Lys Thr Pro Pro Ser Pro Gly Gln Ala Val Glu Cys Cys Gln
50 55 60 Gly Asp Trp Cys Asn Arg Asn Ile Thr Ala Gln Leu 65 70 75 8
81 PRT Homo sapiens 8 Phe Leu Lys Cys Tyr Cys Ser Gly His Cys Pro
Asp Asp Ala Ile Asn 1 5 10 15 Asn Thr Cys Ile Thr Asn Gly His Cys
Phe Ala Ile Ile Glu Glu Asp 20 25 30 Asp Gln Gly Glu Thr Thr Leu
Ala Ser Gly Cys Met Lys Tyr Glu Gly 35 40 45 Ser Asp Phe Gln Cys
Lys Asp Ser Pro Lys Ala Gln Leu Arg Arg Thr 50 55 60 Ile Glu Cys
Cys Arg Thr Asn Leu Cys Asn Gln Tyr Leu Gln Pro Thr 65 70 75 80 Leu
9 82 PRT Homo sapiens 9 Val Leu Arg Cys Lys Cys His His His Cys Pro
Glu Asp Ser Val Asn 1 5 10 15 Asn Ile Cys Ser Thr Asp Gly Tyr Cys
Phe Thr Met Ile Glu Glu Asp 20 25 30 Asp Ser Gly Leu Pro Val Val
Thr Ser Gly Cys Leu Gly Leu Glu Gly 35 40 45 Ser Asp Phe Gln Cys
Arg Asp Thr Pro Ile Pro His Gln Arg Arg Ser 50 55 60 Ile Glu Cys
Cys Thr Glu Arg Asn Glu Cys Asn Lys Asp Leu His Pro 65 70 75 80 Thr
Leu 10 93 PRT Homo sapiens 10 Glu Thr Gln Glu Cys Leu Phe Phe Asn
Ala Asn Trp Glu Lys Asp Arg 1 5 10 15 Thr Asn Gln Thr Gly Val Glu
Pro Cys Tyr Gly Asp Lys Asp Lys Arg 20 25 30 Arg His Cys Phe Ala
Thr Trp Lys Asn Ile Ser Gly Ser Ile Glu Ile 35 40 45 Val Lys Gln
Gly Cys Trp Leu Asp Asp Ile Asn Cys Tyr Asp Arg Thr 50 55 60 Asp
Cys Val Glu Lys Lys Asp Ser Pro Glu Val Tyr Phe Cys Cys Cys 65 70
75 80 Glu Gly Asn Met Cys Asn Glu Lys Phe Ser Tyr Phe Pro 85 90 11
93 PRT Homo sapiens 11 Glu Thr Arg Glu Cys Ile Tyr Tyr Asn Ala Asn
Trp Glu Leu Glu Arg 1 5 10 15 Thr Asn Gln Ser Gly Leu Glu Arg Cys
Glu Gly Glu Gln Asp Lys Arg 20 25 30 Leu His Cys Tyr Ala Ser Trp
Arg Asn Ser Ser Gly Thr Ile Glu Leu 35 40 45 Val Lys Lys Gly Cys
Trp Leu Asp Asp Phe Asn Cys Tyr Asp Arg Gln 50 55 60 Glu Cys Val
Ala Thr Glu Glu Asn Pro Gln Val Tyr Phe Cys Cys Cys 65 70 75 80 Glu
Gly Asn Phe Cys Asn Glu Arg Phe Thr His Leu Pro 85 90 12 96 PRT
Homo sapiens 12 Phe Lys Asp Pro Tyr Gln Gln Asp Leu Gly Ile Gly Glu
Ser Arg Ile 1 5 10 15 Ser His Glu Asn Gly Thr Ile Leu Cys Ser Lys
Gly Ser Thr Cys Tyr 20 25 30 Gly Leu Trp Glu Lys Ser Lys Gly Asp
Ile Asn Leu Val Lys Gln Gly 35 40 45 Cys Trp Ser His Ile Gly Asp
Pro Gln Glu Cys His Tyr Glu Glu Cys 50 55 60 Val Val Thr Thr Thr
Pro Pro Ser Ile Gln Asn Gly Thr Tyr Arg Phe 65 70 75 80 Cys Cys Cys
Ser Thr Asp Leu Cys Asn Val Asn Phe Thr Glu Asn Phe 85 90 95 13 184
PRT Homo sapiens 13 Met Ser Arg Thr Ala Tyr Thr Val Gly Ala Leu Leu
Leu Leu Leu Gly 1 5 10 15 Thr Leu Leu Pro Ala Ala Glu Gly Lys Lys
Lys Gly Ser Gln Gly Ala 20 25 30 Ile Pro Pro Pro Asp Lys Ala Gln
His Asn Asp Ser Glu Gln Thr Gln 35 40 45 Ser Pro Gln Gln Pro Gly
Ser Arg Asn Arg Gly Arg Gly Gln Gly Arg 50 55 60 Gly Thr Ala Met
Pro Gly Glu Glu Val Leu Glu Ser Ser Gln Glu Ala 65 70 75 80 Leu His
Val Thr Glu Arg Lys Tyr Leu Lys Arg Asp Trp Cys Lys Thr 85 90 95
Gln Pro Leu Lys Gln Thr Ile His Glu Glu Gly Cys Asn Ser Arg Thr 100
105 110 Ile Ile Asn Arg Phe Cys Tyr Gly Gln Cys Asn Ser Phe Tyr Ile
Pro 115 120 125 Arg His Ile Arg Lys Glu Glu Gly Ser Phe Gln Ser Cys
Ser Phe Cys 130 135 140 Lys Pro Lys Lys Phe Thr Thr Met Met Val Thr
Leu Asn Cys Pro Glu 145 150 155 160 Leu Gln Pro Pro Thr Lys Lys Lys
Arg Val Thr Arg Val Lys Gln Cys 165 170 175 Arg Cys Ile Ser Ile Asp
Leu Asp 180 14 232 PRT Homo sapiens 14 Met Glu Arg Cys Pro Ser Leu
Gly Val Thr Leu Tyr Ala Leu Val Val 1 5 10 15 Val Leu Gly Leu Arg
Ala Thr Pro Ala Gly Gly Gln His Tyr Leu His 20 25 30 Ile Arg Pro
Ala Pro Ser Asp Asn Leu Pro Leu Val Asp Leu Ile Glu 35 40 45 His
Pro Asp Pro Ile Phe Asp Pro Lys Glu Lys Asp Leu Asn Glu Thr 50 55
60 Leu Leu Arg Ser Leu Leu Gly Gly His Tyr Asp Pro Gly Phe Met Ala
65 70 75 80 Thr Ser Pro Pro Glu Asp Arg Pro Gly Gly Gly Gly Gly Ala
Ala Gly 85 90 95 Gly Ala Glu Asp Leu Ala Glu Leu Asp Gln Leu Leu
Arg Gln Arg Pro 100 105 110 Ser Gly Ala Met Pro Ser Glu Ile Lys Gly
Leu Glu Phe Ser Glu Gly 115 120 125 Leu Ala Gln Gly Lys Lys Gln Arg
Leu Ser Lys Lys Leu Arg Arg Lys 130 135 140 Leu Gln Met Trp Leu Trp
Ser Gln Thr Phe Cys Pro Val Leu Tyr Ala 145 150 155 160 Trp Asn Asp
Leu Gly Ser Arg Phe Trp Pro Arg Tyr Val Lys Val Gly 165 170 175 Ser
Cys Phe Ser Lys Arg Ser Cys Ser Val Pro Glu Gly Met Val Cys 180 185
190 Lys Pro Ser Lys Ser Val His Leu Thr Val Leu Arg Trp Arg Cys Gln
195 200 205 Arg Arg Gly Gly Gln Arg Cys Gly Trp Ile Pro Ile Gln Tyr
Pro Ile 210 215 220 Ile Ser Glu Cys Lys Cys Ser Cys 225 230 15 139
PRT Artificial Synthetic 15 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn
Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Xaa Arg Met Ala
Ser 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 Asp 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 16 139 PRT Artificial Synthetic 16 Ser Thr Gly Gly Lys Gln
Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala
Leu Arg Xaa Ala Ser 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 Asp 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 17 139 PRT Artificial Synthetic 17 Ser Thr
Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15
Asn Gln Glu Ala Leu Arg Met Ala Ser Xaa 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 Asp 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 18 139 PRT Artificial
Synthetic 18 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys
Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser Val Ala
Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Xaa 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 Asp
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 19
139 PRT Artificial Synthetic 19 Ser Thr Gly Gly Lys Gln Arg Ser Gln
Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met
Ala Ser Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg Gln Xaa
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 Asp 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 20 139 PRT Artificial Synthetic 20 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser 20 25 30 Asp Gln Arg
Gln Ala Cys Xaa 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 Asp 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 21 139 PRT Artificial Synthetic 21 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser 20 25
30 Asp Gln Arg Gln Ala Cys Lys Lys His Xaa 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 Asp 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 22 139 PRT Artificial Synthetic
22 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser
Ser Ser 20 25 30 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Xaa
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 Asp 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 23 139 PRT
Artificial Synthetic 23 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Xaa 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 Asp 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 24 139 PRT Artificial Synthetic 24 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Xaa
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 Asp 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 25 139 PRT Artificial Synthetic 25 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa 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 Asp 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 26 139 PRT Artificial Synthetic
26 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa 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 Asp 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 27 139 PRT
Artificial Synthetic 27 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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
Xaa 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 Asp 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 28 139 PRT Artificial Synthetic 28 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Xaa 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 Asp 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 29 139 PRT Artificial Synthetic 29 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa 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 Asp 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 30 139 PRT Artificial Synthetic
30 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa 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 Asp 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 31 139 PRT
Artificial Synthetic 31 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Xaa 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 Asp 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 32 139 PRT Artificial Synthetic 32 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Xaa 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 Asp 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 33 139 PRT Artificial Synthetic 33 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa 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 Asp 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 34 139 PRT Artificial Synthetic
34 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa
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 Asp 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 35 139 PRT
Artificial Synthetic 35 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Xaa 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 Asp 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 36 139 PRT Artificial Synthetic 36 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Xaa 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 Asp 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 37 139 PRT Artificial Synthetic 37 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn
Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala
Ser 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 Xaa 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 Asp 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 38 139 PRT Artificial Synthetic 38 Ser Thr Gly Gly Lys Gln
Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala
Leu Arg Met Ala Ser 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 Xaa 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 Asp 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 39 139 PRT Artificial Synthetic 39 Ser Thr
Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15
Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa 65 70 75 80 Ala Thr Asn His Ala Ile Val Gln Thr
Leu Val His Phe Ile Asn Pro 85 90 95 Asp 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 40 139 PRT Artificial
Synthetic 40 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys
Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa Asn
His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 95 Asp
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 41
139 PRT Artificial Synthetic 41 Ser Thr Gly Gly Lys Gln Arg Ser Gln
Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met
Ala Ser 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 Xaa His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn
Pro 85 90 95 Asp 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 42 139 PRT Artificial Synthetic 42 Ser Thr Gly Gly
Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln
Glu Ala Leu Arg Met Ala Ser 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 Xaa Val Gln Thr Leu Val
His Phe Ile Asn Pro 85 90 95 Asp 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 43 139 PRT Artificial Synthetic 43
Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5
10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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
Xaa Thr Leu Val His Phe Ile Asn Pro 85 90 95 Asp 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 44 139 PRT
Artificial Synthetic 44 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Xaa Val His Phe Ile Asn Pro 85 90
95 Asp 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 45 139 PRT Artificial Synthetic 45 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Xaa Ile
Asn Pro 85 90 95 Asp 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 46 139 PRT Artificial Synthetic 46 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa Asn Pro 85 90 95 Asp 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 47 139 PRT Artificial Synthetic
47 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Xaa Pro 85 90 95 Asp 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 48 139 PRT
Artificial Synthetic 48 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Xaa 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 49 139 PRT Artificial Synthetic 49 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Asp Xaa 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 50 139 PRT Artificial Synthetic 50 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Asp Thr Val Pro Lys Pro Cys Cys
Xaa 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 51 139 PRT Artificial Synthetic
51 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Asp Thr Val Pro
Lys Pro Cys Cys Ala Pro Thr Xaa 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 52 139 PRT
Artificial Synthetic 52 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Xaa 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 53 139 PRT Artificial Synthetic 53 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln
Leu Asn Xaa 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 54 139 PRT Artificial Synthetic 54 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Asp Thr Val Pro Lys Pro Cys Cys
Ala Pro Thr Gln Leu Asn Ala Ile 100 105 110 Ser Val Xaa 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 55 139 PRT Artificial Synthetic
55 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Asp Thr Val Pro
Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile 100 105 110 Ser Val
Leu Xaa 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 56 139 PRT
Artificial Synthetic 56 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile
100 105 110 Ser Val Leu Tyr Xaa 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 57 139 PRT Artificial Synthetic 57 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln
Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Xaa 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 58 139 PRT Artificial Synthetic 58 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Asp 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 Xaa Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val
Val Arg Ala Cys Gly Cys His 130 135 59 139 PRT Artificial Synthetic
59 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Asp 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 Xaa Asn Val Ile Leu Lys Lys Tyr 115 120 125
Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 60 139 PRT
Artificial Synthetic 60 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Asp 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 Xaa Val Ile Leu Lys
Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130
135 61 139 PRT Artificial Synthetic 61 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Asp 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
Xaa Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys
Gly Cys His 130 135 62 139 PRT Artificial Synthetic 62 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Asp 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 Xaa Lys Lys Tyr 115 120 125 Arg Asn Met Val
Val Arg Ala Cys Gly Cys His 130 135 63 139 PRT Artificial Synthetic
63 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Asp 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 Xaa Lys Tyr 115 120 125
Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 64 139 PRT
Artificial Synthetic 64 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Asp 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
Xaa Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130
135 65 139 PRT Artificial Synthetic 65 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Asp 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 Xaa 115 120 125 Arg Asn Met Val Val Arg Ala Cys
Gly Cys His 130 135 66 139 PRT Artificial Synthetic 66 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Asp 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 Xaa Asn Met Val
Val Arg Ala Cys Gly Cys His 130 135 67 139 PRT Artificial Synthetic
67 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys
1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser 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 Asp 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 Xaa Met Val Val Arg Ala Cys Gly Cys His 130 135 68 139 PRT
Artificial Synthetic 68 Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg
Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu Arg Met Ala Ser
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 Asp 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 Xaa Ala Cys Gly Cys His 130
135 69 139 PRT Artificial Synthetic 69 Ser Thr Gly Gly Lys Gln Arg
Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn Gln Glu Ala Leu
Arg Met Ala Ser 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 Asp 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 Xaa Cys
Gly Cys His 130 135 70 139 PRT Artificial Synthetic 70 Ser Thr Gly
Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 15 Asn
Gln Glu Ala Leu Arg Met Ala Ser 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 Asp 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 Xaa 130 135 71 103 PRT Artificial Synthetic
71 Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe Xaa Asp Xaa Gly Trp Xaa
1 5 10 15 Asp Trp Ile Ile Ala Pro Xaa Gly Tyr Xaa Ala Xaa Tyr Cys
Xaa Gly 20 25 30 Xaa Cys Xaa Phe Pro Leu Xaa Xaa Xaa Xaa Asn Xaa
Thr Asn His Ala 35 40 45 Ile Xaa Gln Thr Leu Val Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Pro 50
55 60 Lys Xaa Cys Cys Xaa Pro Thr Xaa Leu Xaa Ala Xaa Ser Xaa Leu
Tyr 65 70 75 80 Xaa Asp Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Tyr Xaa
Xaa Met Xaa 85 90 95 Val Xaa Xaa Cys Gly Cys Xaa 100 72 103 PRT
Homo sapiens 72 Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile
Gly Trp Ser 1 5 10 15 Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala
Tyr Tyr Cys Ser Gly 20 25 30 Ala Cys Gln Phe Pro Met Pro Lys Ser
Leu Lys Pro Ser Asn His Ala 35 40 45 Thr Ile Gln Ser Ile Val Arg
Ala Val Gly Val Val Pro Gly Ile Pro 50 55 60 Glu Pro Cys Cys Val
Pro Glu Lys Met Ser Ser Leu Ser Ile Leu Phe 65 70 75 80 Phe Asp Glu
Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met Thr 85 90 95 Val
Glu Ser Cys Ala Cys Arg 100 73 103 PRT Homo sapiens 73 Cys Ser Arg
Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp Asn 1 5 10 15 Glu
Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ala Gly 20 25
30 Ala Cys Glu Phe Pro Met Pro Lys Ile Val Arg Pro Ser Asn His Ala
35 40 45 Thr Ile Gln Ser Ile Val Arg Ala Val Gly Ile Ile Pro Gly
Ile Pro 50 55 60 Glu Pro Cys Cys Val Pro Asp Lys Met Asn Ser Leu
Gly Val Leu Phe 65 70 75 80 Leu Asp Glu Asn Arg Asn Val Val Leu Lys
Val Tyr Pro Asn Met Ser 85 90 95 Val Asp Thr Cys Ala Cys Arg 100 74
103 PRT Homo sapiens 74 Cys Gln Lys Thr Ser Leu Arg Val Asn Phe Glu
Asp Ile Gly Trp Asp 1 5 10 15 Ser Trp Ile Ile Ala Pro Lys Glu Tyr
Glu Ala Tyr Glu Cys Lys Gly 20 25 30 Gly Cys Phe Phe Pro Leu Ala
Asp Asp Val Thr Pro Thr Lys His Ala 35 40 45 Ile Val Gln Thr Leu
Val His Leu Lys Phe Pro Thr Lys Val Gly Lys 50 55 60 Ala Cys Cys
Val Pro Thr Lys Leu Ser Pro Ile Ser Val Leu Tyr Lys 65 70 75 80 Asp
Asp Met Gly Val Pro Thr Leu Lys Tyr His Tyr Glu Gly Met Ser 85 90
95 Val Ala Glu Cys Gly Cys Arg 100 75 102 PRT Homo sapiens 75 Cys
Lys Arg Thr Pro Leu Tyr Ile Asp Phe Lys Glu Ile Gly Trp Asp 1 5 10
15 Ser Trp Ile Ile Ala Pro Pro Gly Tyr Glu Ala Tyr Glu Cys Arg Gly
20 25 30 Val Cys Asn Tyr Pro Leu Ala Glu His Leu Thr Pro Thr Lys
His Ala 35 40 45 Ile Ile Gln Ala Leu Val His Leu Lys Asn Ser Gln
Lys Ala Ser Lys 50 55 60 Ala Cys Cys Val Pro Thr Lys Leu Glu Pro
Ile Ser Ile Leu Tyr Leu 65 70 75 80 Asp Lys Gly Val Val Thr Tyr Lys
Phe Lys Tyr Glu Gly Met Ala Val 85 90 95 Ser Glu Cys Gly Cys Arg
100 76 106 PRT Homo sapiens 76 Cys Arg Ala Arg Arg Leu Tyr Val Ser
Phe Arg Glu Val Gly Trp His 1 5 10 15 Arg Trp Val Ile Ala Pro Arg
Gly Phe Leu Ala Asn Tyr Cys Gln Gly 20 25 30 Gln Cys Ala Leu Pro
Val Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala 35 40 45 Leu Asn His
Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro Gly 50 55 60 Ala
Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro Ile Ser 65 70
75 80 Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gln Tyr
Glu 85 90 95 Asp Met Val Val Asp Glu Cys Gly Cys Arg 100 105 77 101
PRT Homo sapiens 77 Cys His Arg His Gln Leu Phe Ile Asn Phe Arg Asp
Leu Gly Trp His 1 5 10 15 Lys Trp Ile Ile Ala Pro Lys Gly Phe Met
Ala Asn Tyr Cys His Gly 20 25 30 Glu Cys Pro Phe Ser Leu Thr Ile
Ser Leu Asn Ser Ser Asn Tyr Ala 35 40 45 Phe Met Gln Ala Leu Met
His Ala Val Asp Pro Glu Ile Pro Gln Ala 50 55 60 Val Cys Ile Pro
Thr Lys Leu Ser Pro Ile Ser Met Leu Tyr Gln Asp 65 70 75 80 Asn Asn
Asp Asn Val Ile Leu Arg His Tyr Glu Asp Met Val Val Asp 85 90 95
Glu Cys Gly Cys Gly 100 78 102 PRT Homo sapiens 78 Cys Ser Arg Lys
Ala Leu His Val Asn Phe Lys Asp Met Gly Trp Asp 1 5 10 15 Asp Trp
Ile Ile Ala Pro Leu Glu Tyr Glu Ala Phe His Cys Glu Gly 20 25 30
Leu Cys Glu Phe Pro Leu Arg Ser His Leu Glu Pro Thr Asn His Ala 35
40 45 Val Ile Gln Thr Leu Met Asn Ser Met Asp Pro Glu Ser Thr Pro
Pro 50 55 60 Thr Cys Cys Val Pro Thr Arg Leu Ser Pro Ile Ser Ile
Leu Phe Ile 65 70 75 80 Asp Ser Ala Asn Asn Val Val Tyr Lys Gln Tyr
Glu Asp Met Val Val 85 90 95 Glu Ser Cys Gly Cys Arg 100 79 102 PRT
Homo sapiens 79 Cys Ser Arg Lys Pro Leu His Val Asp Phe Lys Glu Leu
Gly Trp Asp 1 5 10 15 Asp Trp Ile Ile Ala Pro Leu Asp Tyr Glu Ala
Tyr His Cys Glu Gly 20 25 30 Leu Cys Asp Phe Pro Leu Arg Ser His
Leu Glu Pro Thr Asn His Ala 35 40 45 Ile Ile Gln Thr Leu Leu Asn
Ser Met Ala Pro Asp Ala Ala Pro Ala 50 55 60 Ser Cys Cys Val Pro
Ala Arg Leu Ser Pro Ile Ser Ile Leu Tyr Ile 65 70 75 80 Asp Ala Ala
Asn Asn Val Val Tyr Lys Gln Tyr Glu Asp Met Val Val 85 90 95 Glu
Ala Cys Gly Cys Arg 100 80 95 PRT Homo sapiens 80 Cys Cys Arg Tyr
Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp 1 5 10 15 Trp Ile
Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Glu 20 25 30
Cys Glu Phe Val Phe Leu Gln Lys Tyr Pro His Thr His Leu Val His 35
40 45 Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr
Lys 50 55 60 Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Gly Lys Glu
Gln Ile Ile 65 70 75 80 Tyr Gly Lys Ile Pro Ala Met Val Val Asp Arg
Cys Gly Cys Ser 85 90 95 81 95 PRT Homo sapiens 81 Cys Cys Arg Tyr
Pro Leu Thr Val Asp Phe Glu Ala Phe Gly Trp Asp 1 5 10 15 Trp Ile
Ile Ala Pro Lys Arg Tyr Lys Ala Asn Tyr Cys Ser Gly Gln 20 25 30
Cys Glu Tyr Met Phe Met Gln Lys Tyr Pro His Thr His Leu Val Gln 35
40 45 Gln Ala Asn Pro Arg Gly Ser Ala Gly Pro Cys Cys Thr Pro Thr
Lys 50 55 60 Met Ser Pro Ile Asn Met Leu Tyr Phe Asn Asp Lys Gln
Gln Ile Ile 65 70 75 80 Tyr Gly Lys Ile Pro Gly Met Val Val Asp Arg
Cys Gly Cys Ser 85 90 95 82 102 PRT Homo sapiens 82 Cys Ser Leu His
Pro Phe Gln Ile Ser Phe Arg Gln Leu Gly Trp Asp 1 5 10 15 His Trp
Ile Ile Ala Pro Pro Phe Tyr Thr Pro Asn Tyr Cys Lys Gly 20 25 30
Thr Cys Leu Arg Val Leu Arg Asp Gly Leu Asn Ser Pro Asn His Ala 35
40 45 Ile Ile Gln Asn Leu Ile Asn Gln Leu Val Asp Gln Ser Val Pro
Arg 50 55 60 Pro Ser Cys Val Pro Tyr Lys Tyr Val Pro Ile Ser Val
Leu Met Ile 65 70 75 80 Glu Ala Asn Gly Ser Ile Leu Tyr Lys Glu Tyr
Glu Gly Met Ile Ala 85 90 95 Glu Ser Cys Thr Cys Arg 100 83 101 PRT
Homo sapiens 83 Cys Arg Lys Val Lys Phe Gln Val Asp Phe Asn Leu Ile
Gly Trp Gly 1 5 10 15 Ser Trp Ile Ile Tyr Pro Lys Gln Tyr Asn Ala
Tyr Arg Cys Glu Gly 20 25 30 Glu Cys Pro Asn Pro Val Gly Glu Glu
Phe His Pro Thr Asn His Ala 35 40 45 Tyr Ile Gln Ser Leu Leu Lys
Arg Tyr Gln Pro His Arg Val Pro Ser 50 55 60 Thr Cys Cys Ala Pro
Val Lys Thr Lys Pro Leu Ser Met Leu Tyr Val 65 70 75 80 Asp Asn Gly
Arg Val Leu Leu Asp His His Lys Asp Met Ile Val Glu 85 90 95 Glu
Cys Gly Cys Leu 100 84 4 PRT Artificial Synthetic 84 Arg Xaa Xaa
Xaa 1 85 12 PRT Homo sapiens 85 Glu Val His Leu Arg Ser Ile Arg Ser
Thr Gly Gly 1 5 10 86 4 PRT Artificial Synthetic 86 Asn Xaa Xaa Xaa
1 87 4 PRT Artificial Synthetic 87 Arg Xaa Xaa Arg 1
* * * * *
References