U.S. patent application number 10/671317 was filed with the patent office on 2005-03-10 for osteogenic devices.
This patent application is currently assigned to Stryker Biotech Corporation. Invention is credited to Kuberasampath, Thangavel, Oppermann, Hermann, Ozkaynak, Engin, Rueger, David C..
Application Number | 20050054825 10/671317 |
Document ID | / |
Family ID | 27391116 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050054825 |
Kind Code |
A1 |
Oppermann, Hermann ; et
al. |
March 10, 2005 |
Osteogenic devices
Abstract
Disclosed are 1) osteogenic devices comprising a matrix
containing osteogenic protein and methods of inducing endochondral
bone growth in mammals using the devices; 2) amino acid sequence
data, amino acid composition, solubility properties, structural
features, homologies and various other data characterizing
osteogenic proteins, 3) methods of producing osteogenic proteins
using recombinant DNA technology, and 4) osteogenically and
chondrogenically active synthetic protein constructs.
Inventors: |
Oppermann, Hermann; (Medway,
MA) ; Kuberasampath, Thangavel; (Medway, MA) ;
Rueger, David C.; (West Roxbury, MA) ; Ozkaynak,
Engin; (Milford, MA) |
Correspondence
Address: |
TESTA, HURWITZ & THIBEAULT, LLP
HIGH STREET TOWER
125 HIGH STREET
BOSTON
MA
02110
US
|
Assignee: |
Stryker Biotech Corporation
Hopkinton
MA
|
Family ID: |
27391116 |
Appl. No.: |
10/671317 |
Filed: |
September 25, 2003 |
Related U.S. Patent Documents
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Application
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Patent Number |
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10671317 |
Sep 25, 2003 |
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09956582 |
Sep 19, 2001 |
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09956582 |
Sep 19, 2001 |
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09074299 |
May 7, 1998 |
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6297213 |
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09074299 |
May 7, 1998 |
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08417071 |
Apr 4, 1995 |
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5814604 |
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08417071 |
Apr 4, 1995 |
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08145812 |
Nov 1, 1993 |
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5750651 |
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08145812 |
Nov 1, 1993 |
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07995345 |
Dec 22, 1992 |
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5258494 |
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07995345 |
Dec 22, 1992 |
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07315342 |
Feb 23, 1989 |
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5011691 |
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07315342 |
Feb 23, 1989 |
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07232630 |
Aug 15, 1988 |
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07232630 |
Aug 15, 1988 |
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07179406 |
Apr 8, 1988 |
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4968590 |
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Current U.S.
Class: |
530/350 ;
424/426 |
Current CPC
Class: |
A61L 27/24 20130101;
C08L 89/06 20130101; A61L 2430/02 20130101; A61C 8/0006 20130101;
A61P 1/02 20180101; C07K 14/51 20130101; A61K 38/00 20130101; A61L
27/34 20130101; A61L 27/365 20130101; A61P 43/00 20180101; A61L
27/3604 20130101; A61L 27/56 20130101; A61L 27/3608 20130101; A61L
27/3654 20130101; A61F 2310/00365 20130101; A61L 27/227 20130101;
A61K 9/0024 20130101; A61L 27/34 20130101 |
Class at
Publication: |
530/350 ;
424/426 |
International
Class: |
C07K 014/47 |
Claims
What is claimed is:
1-80. (canceled)
81. An osteogenic protein comprising one or more polypeptide chains
capable of inducing endochondral bone formation when disposed
within a matrix and implanted in a mammal, wherein said polyeptide
chain is further characterized as having cysteine residues in the
same relative positions as the cysteine skeleton sequence:
21 1 10 20 30 40 XXXXXXXXXXXXXXXXXXXXXXXXCXXXCXXXXX 50 60 70
XXXXXXXXXXXXXXXXXXXXXXXXXXCCXXXXXX 80 90 100
XXXXXXXXXXXXXXXXXXXXXXXXXCXCX,
or a point mutation thereof, wherein said protein or said mutant
protein is capable of inducing endochondral bone formation in a
mammal, and wherein each X represents any amino acid.
82. An osteogenic protein comprising one or more polypeptide chains
capable of inducing endochondral bone formation when disposed
within a matrix and implanted in a mammal, wherein said polypeptide
chain is further characterized as having cysteine residues in the
same relative positions as the cysteine skeleton sequence:
22 1 10 20 30 40 CXXXXXXXXXXXXXXXXXXXXXXXXXXXXCXXXCXXXXXX 50 60 70
80 XXXXXXXXXXXXXXXXXXXXXXXXXCCXXXXXXXXXXX- XX 90 100
XXXXXXXXXXXXXXXXXXCXCX,
or a point mutation thereof, wherein said protein or said mutant
protein is capable of inducing endochondral bone formation in a
mammal, and wherein each X represents any amino acid.
83. A protein, produced by expression of recombinant DNA in a host
cell, comprising one or more polypeptide chains having a
conformation competent to induce bone formation when combined with
a matrix and implanted in a mammal, said polypeptide chain having
at least 96 amino acids and less than about 200 amino acids, and
having a molecular weight of approximately 14-16 kDa in an
unglycosylated form or a molecular weight of approximately 16-18
kDa in a glycosylated form as determined by polyacrylamide gel
electrophoresis under reducing conditions, wherein said polypeptide
chain is encoded by a DNA, one strand of which hybridizes
selectively to:
23 10 20 30 40 50
GATCCTAATGGGCTGTACGTGGACTTCCAGCGCGACGTGGGCTGGGACGA D P N G L Y V D
F Q R D V G W D D 60 70 80 90 100
CTGGATCATCGCCCCCGTCGACTTCGACGCCTACTACT- GCTCCGGAGCCT W I I A P V D
F D A Y Y C S G A 110 120 130 140 150
GCCAGTTCCCCTCTGCGGATCACTTCAACAGCACCAACCACGCCGTGGTG C Q F P S A D H
F N S T N H A V V 160 170 180 190 200
CAGACCCTGGTGAACAACATGAACCCCGGCAAGGTACC- CAAGCCCTGCTG Q T L V N N M
N P G K V P K P C C 210 220 230 240 250
CGTGCCCACCGAGCTGTCCGCCATCAGCATGCTGTACCTGGACGAGAATT V P T E L S A I
S M L Y L D E N 260 270 280 290 300
CCACCGTGGTGCTGAAGAACTACCAGGAGATGACCGTGGT- GGGCTGCGGC S T V V L K N
Y Q E M T V V G C G 310 TGCCGCTAACTGCAG, C R
in 5.times.SSPE, 10.times. Denhardt's mix, 0.5% SDS at 50.degree.
C., and further wherein said polypeptide chain is further
characterized as having cysteine residues in the same relative
positions as the cysteine skeleton sequence:
24 1 10 20 30 40 XXXXXXXXXXXXXXXXXXXXXXXXCXXXCXXXXX 50 60 70
XXXXXXXXXXXXXXXXXXXXXXXXXXCCXXXXXX 80 90 100
XXXXXXXXXXXXXXXXXXXXXXXXXCXCX,
or a point mutation thereof, wherein said protein or said mutant
protein is capable of inducing endochondral bone formation in a
mammal, and wherein each said X is an amino acid.
84. The protein of claim 83, wherein said polypeptide chain is
further characterized as having cysteine residues in the same
relative positions as the cysteine skeleton sequence:
25 1 10 20 30 40 CXXXXXXXXXXXXXXXXXXXXXXXXXXXXCXXXCXXXXXX 50 60 70
80 XXXXXXXXXXXXXXXXXXXXXXXXXCCXXXXXXXXXXX- XX 90 100
XXXXXXXXXXXXXXXXXXCXCX,
or a point mutation thereof, wherein said protein or said mutant
protein is capable of inducing endochondral bone formation in a
mammal, and wherein each said X is an amino acid.
85. The osteogenic protein of any one of claims 81-84, wherein said
protein is a dimeric protein.
86. The osteogenic protein of any one of claims 81-84, wherein said
protein is glycosylated.
87. The osteogenic protein of any one of claims 81-84, wherein said
protein is unglycosylated.
88. A device for implantation in a mammal, comprising: a
biocompatible, in vivo biodegradable matrix defining pores of a
dimension sufficient to permit influx, proliferation and
differentiation of migratory progenitor cells from the body of said
mammal; and a substantially pure osteogenic protein comprising one
or more polypeptide chains capable of inducing endochondral bone
formation when disposed within a matrix and implanted in a mammal,
wherein said polypeptide chain is further characterized as having
cysteine residues in the same relative positions as the cysteine
skeleton sequence:
26 1 10 20 30 40 XXXXXXXXXXXXXXXXXXXXXXXXCXXXCXXXXX 50 60 70
XXXXXXXXXXXXXXXXXXXXXXXXXXCCXXXXXX 80 90 100
XXXXXXXXXXXXXXXXXXXXXXXXXCXCX,
or a point mutation thereof, wherein said protein or said mutant
protein is capable of inducing endochondral bone formation in a
mammal, and wherein each X represents any amino acid.
89. A device for implantation in a mammal, comprising: a
biocompatible, in vivo biodegradable matrix defining pores of a
dimension sufficient to permit influx, proliferation and
differentiation of migratory progenitor cells from the body of said
mammal; and a substantially pure osteogenic protein comprising one
or more polypeptide chains capable of inducing endochondral bone
formation when disposed within a matrix and implanted in a mammal,
wherein said polypeptide chain is further characterized as having
cysteine residues in the same relative positions as the cysteine
skeleton sequence:
27 1 10 20 30 40 CXXXXXXXXXXXXXXXXXXXXXXXXXXXXCXXXCXXXXXX 50 60 70
80 XXXXXXXXXXXXXXXXXXXXXXXXXCCXXXXXXXXXXX- XX 90 100
XXXXXXXXXXXXXXXXXXCXCX,
or a point mutation thereof, wherein said protein or said mutant
protein is capable of inducing endochondral bone formation in a
mammal, and wherein each X represents any amino acid.
90. A device for implantation in a mammal, comprising: a
biocompatible, in vivo biodegradable matrix defining pores of a
dimension sufficient to permit influx, proliferation and
differentiation of migratory progenitor cells from the body of said
mammal; and a substantially pure protein, produced by expression of
recombinant DNA in a host cell, comprising one or more polypeptide
chains having a conformation competent to induce bone formation
when combined with a matrix and implanted in a mammal, said
polypeptide chain having at least 96 amino acids and less than
about 200 amino acids, and having a molecular weight of
approximately 14-16 kDa in an unglycosylated form or a molecular
weight of approximately 16-18 kDa in a glycosylated form as
determined by polyacrylamide gel electrophoresis under reducing
conditions, wherein said polypeptide chain is encoded by a DNA, one
strand of which hybridizes selectively to:
28 10 20 30 40 50
GATCCTAATGGGCTGTACGTGGACTTCCAGCGCGACGTGGGCTGGGACGA D P N G L Y V D
F Q R D V G W D D 60 70 80 90 100
CTGGATCATCGCCCCCGTCGACTTCGACGCCTACTACT- GCTCCGGAGCCT W I I A P V D
F D A Y Y C S G A 110 120 130 140 150
GCCAGTTCCCCTCTGCGGATCACTTCAACAGCACCAACCACGCCGTGGTG C Q F P S A D H
F N S T N H A V V 160 170 180 190 200
CAGACCCTGGTGAACAACATGAACCCCGGCAAGGTACC- CAAGCCCTGCTG Q T L V N N M
N P G K V P K P C C 210 220 230 240 250
CGTGCCCACCGAGCTGTCCGCCATCAGCATGCTGTACCTGGACGAGAATT V P T E L S A I
S M L Y L D E N 260 270 280 290 300
CCACCGTGGTGCTGAAGAACTACCAGGAGATGACCGTGGT- GGGCTGCGGC S T V V L K N
Y Q E M T V V G C G 310 TGCCGCTAACTGCAG, C R
in 5.times.SSPE, 10.times. Denhardt's mix, 0.5% SDS at 50.degree.
C., and further wherein said polypeptide chain is further
characterized as having cysteine residues in the same relative
positions as the cysteine skeleton sequence:
29 1 10 20 30 40 XXXXXXXXXXXXXXXXXXXXXXXXCXXXCXXXXX 50 60 70
XXXXXXXXXXXXXXXXXXXXXXXXXXCCXXXXXX 80 90 100
XXXXXXXXXXXXXXXXXXXXXXXXXCXCX,
or a point mutation thereof, wherein said protein or said mutant
protein is capable of inducing endochondral bone formation in a
mammal, and wherein each said X is an amino acid.
91. The device of claim 90, wherein said polypeptide chain is
further characterized as having cysteine residues in the same
relative positions as the cysteine skeleton sequence:
30 1 10 20 30 40 CXXXXXXXXXXXXXXXXXXXXXXXXXXXXCXXXCXXXXXX 50 60 70
80 XXXXXXXXXXXXXXXXXXXXXXXXXCCXXXXXXXXXXX- XX 90 100
XXXXXXXXXXXXXXXXXXCXCX,
or a point mutation thereof, wherein said protein or said mutant
protein is capable of inducing endochondral bone formation in a
mammal, and wherein each said X is an amino acid.
92. The device of any one of claims 88-91, wherein said matrix
comprises collagen and at least one material selected from the
group consisting of polymers comprising lactic acid monomer units,
polymers comprising glycolic acid monomer units, bone,
hydroxyapatite, calcium phosphate, muscle, and tissue.
93. The device of any one of claims 88-92, wherein said protein is
a dimeric protein.
94. The device of any one of claims 88-92, wherein said polypeptide
is glycosylated.
95. The device of any one of claims 88-92, wherein said polypeptide
is unglycosylated.
96. A method of inducing endochondral bone formation in a mammal
comprising the step of implanting the device of any one of claims
88-95 in said mammal at a locus accessible to migratory progenitor
cells of said mammal.
97. A method of inducing cartilage formation in a mammal comprising
the step of implanting the device of any one of claims 88-95 in
said mammal at a locus accessible to migratory progenitor cells of
said mammal.
Description
[0001] This application is a continuation-in-part of copending
application Serial Number 232,630, entitled "Osteogenic Devices",
filed Aug. 15, 1988, which is a continuation-in-part of copending
application Serial Number 179,406, entitled "Osteogenic Devices",
filed April 8, 1988.
BACKGROUND OF THE INVENTION
[0002] This invention relates to osteogenic devices, to genes
encoding proteins which can induce osteogenesis in mammals and
methods for their production using recombinant DNA techniques, to
synthetic forms of osteogenic protein, to a method of reproducibly
purifying osteogenic protein from mammalian bone, to matrix
materials which act as a carrier to induce osteogenesis in mammals,
and to bone and cartilage repair procedures using the osteogenic
device.
[0003] Mammalian bone tissue is known to contain one or more
proteinaceous materials, presumably active during growth and
natural bone healing, which can induce a developmental cascade of
cellular events resulting in endochondral bone formation. This
active factor (or factors) has variously been referred to in the
literature as bone morphogenetic or morphogenic protein, bone
inductive protein, osteogenic protein, osteogenin, or
osteoinductive protein.
[0004] The developmental cascade of bone differentiation consists
of chemotaxis of mesenchymal cells, proliferation of progenitor
cells, differentiation of cartilage, vascular invasion, bone
formation, remodeling, and finally marrow differentiation (Reddi
(1981) Collagen Rel. Res. 1:209-226).
[0005] Though the precise mechanisms underlying these phenotypic
transformations are unclear, it has been shown that the natural
endochondral bone differentiation activity of bone matrix can be
dissociatively extracted and reconstituted with inactive residual
collagenous matrix to restore full bone induction activity (Sampath
and Reddi, (1981) Proc. Natl. Acad. Sci. USA 78:7599-7603). This
provides an experimental method for assaying protein extracts for
their ability to induce endochondral bone in vivo.
[0006] This putative bone inductive protein has been shown to have
a molecular mass of less than 50 kilodaltons (kD). Several species
of mammals produce closely related protein as demonstrated by cross
species implant experiments (Sampath and Reddi (1983) Proc. Natl.
Acad. Sci. USA 80:6591-6595).
[0007] The potential utility of these proteins has been widely
recognized. It is contemplated that the availability of the pure
protein would revolutionize orthopedic medicine, certain types of
plastic surgery, and various periodontal and craniofacial
reconstructive procedures.
[0008] The observed properties of these protein fractions have
induced an intense research effort in various laboratories directed
to isolating and identifying the pure factor or factors responsible
for osteogenic activity. The current state of the art of
purification of osteogenic protein from mammalian bone is disclosed
by Sampath et al. (Proc. Natl. Acad. Sci. USA (1987) 80). Urist et
al. (Proc. Soc. Exp. Biol. Med. (1984) 173:194-199) disclose a
human osteogenic protein fraction which was extracted from
demineralized cortical bone by means of a calcium chloride-urea
inorganic-organic solvent mixture, and retrieved by differential
precipitation in guanidine-hydrochloride and preparative gel
electrophoresis. The authors report that the protein fraction has
an amino acid composition of an acidic polypeptide and a molecular
weight in a range of 17-18 kD.
[0009] Urist et al. (Proc. Natl. Acad. Sci. USA (1984) 81:371-375)
disclose a bovine bone morphogenetic protein extract having the
properties of an acidic polypeptide and a molecular weight of
approximately 18 kD. The authors reported that the protein was
present in a fraction separated by hydroxyapatite chromatography,
and that it induced bone formation in mouse hindquarter muscle and
bone regeneration in trephine defects in rat and dog skulls. Their
method of obtaining the extract from bone results in ill-defined
and impure preparations.
[0010] European Patent Application Serial No. 148,155, published
Oct. 7, 1985, purports to disclose osteogenic proteins derived from
bovine, porcine, and human origin. One of the proteins, designated
by the inventors as a P3 protein having a molecular weight of 22-24
kD, is said to have been purified to an essentially homogeneous
state. This material is reported to induce bone formation when
implanted into animals.
[0011] International Application No. PCT/087/01537, published Jan.
14, 1988, discloses an impure fraction from bovine bone which has
bone induction qualities. The named applicants also disclose
putative bone inductive factors produced by recombinant DNA
techniques. Four DNA sequences were retrieved from human or bovine
genomic or cDNA libraries and apparently expressed in recombinant
host cells. While the applicants stated that the expressed proteins
may be bone morphogenic proteins, bone induction was not
demonstrated, suggesting that the recombinant proteins are not
osteogenic. See also Urist et al., EP 0,212,474 entitled Bone
Morphogenic Agents.
[0012] Wang et al. (Proc. Nat. Acad. Sci. USA (1988) 85: 9484-9488)
discloses the purification of a bovine bone morphogenetic protein
from guanidine extracts of demineralized bone having cartilage and
bone formation activity as a basic protein corresponding to a
molecular weight of 30 kD determined from gel elution. Purification
of the protein yielded proteins of 30, 18 and 16 kD which, upon
separation, were inactive. In view of this result, the authors
acknowledged that the exact identity of the active material had not
been determined.
[0013] Wozney et al. (Science (1988) 242: 1528-1534) discloses the
isolation of full-length cDNA's encoding the human equivalents of
three polypeptides originally purified from bovine bone. The
authors report that each of the three recombinantly expressed human
proteins are independently or in combination capable of inducing
cartilage formation. No evidence of bone formation is reported.
[0014] It is an object of this invention to provide osteogenic
devices comprising matrices containing dispersed osteogenic protein
capable of bone induction in allogenic and xenogenic implants.
Another object is to provide a reproducible method of isolating
osteogenic protein from mammalian bone tissue. Another object is to
characterize the protein responsible for osteogenesis. Another
object is to provide natural and recombinant osteogenic proteins
capable of inducing endochondral bone formation in mammals,
including humans. Yet another object is to provide genes encoding
native and non-native osteogenic proteins and methods for their
production using recombinant DNA techniques. Another object is to
provide novel biosynthetic forms of osteogenic proteins and a
structural design for novel, functional osteogenic proteins.
Another object is to provide a suitable deglycosylated collagenous
bone matrix as a carrier for osteogenic protein for use in
xenogenic implants. Another object is to provide methods for
inducing cartilage formation.
[0015] These and other objects and features of the invention will
be apparent from the description, drawings, and claims which
follow.
SUMMARY OF THE INVENTION
[0016] This invention involves osteogenic devices which, when
implanted in a mammalian body, can induce at the locus of the
implant the full developmental cascade of endochondral bone
formation and bone marrow differentiation. Suitably modified as
disclosed herein, the devices also may be used to induce cartilage
formation. The devices comprise a carrier material, referred to
herein as a matrix, having the characteristics disclosed below,
containing dispersed osteogenic protein either in its native form
or in the form of a biosynthetic construct.
[0017] A key to these developments was the elucidation of amino
acid sequence and structure data of native osteogenic protein. A
protocol was developed which results in retrieval of active,
substantially pure osteogenic protein from mammalian bone.
Investigation of the properties and structure of the native form
osteogenic protein then permitted the inventors to develop a
rational design for non-native forms, i.e., forms never before
known in nature, capable of inducing bone formation. As far as
applicants are aware, the constructs disclosed herein constitute
the first instance of the design of a functional, active protein
without preexisting knowledge of the active region of a native form
nucleotide or amino acid sequence.
[0018] A series of consensus DNA sequences were designed with the
goal of producing an active osteogenic protein. The sequences were
based on partial amino acid sequence data obtained from the natural
source product and on observed homologies with unrelated genes
reported in the literature, or the sequences they encode, having a
presumed or demonstrated developmental function. Several of the
biosynthetic consensus sequences have been expressed as fusion
proteins in procaryotes, purified, cleaved, refolded, combined with
a matrix, implanted in an established animal model, and shown to
have endochondral bone-inducing activity. The currently preferred
active totally biosynthetic proteins comprise two synthetic
sequences designated COP5 and COP7. The amino acid sequences of
these proteins are set forth below.
1 1 10 20 30 40 COP5 LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD 50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR 1 10 20 30 40 COP7
LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFP- LAD 50 60 70
HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
[0019] In these sequences and all other amino acid sequences
disclosed herein, the dashes (-) are used as fillers only to line
up comparable sequences in related proteins, and have no other
function. Thus, amino acids 45-50 of COP7, for example, are NHAVV.
Also, the numbering of amino acids is selected solely for purposes
of facilitating comparisons between sequences. Thus, for example,
the DF residues numbered at 9 and 10 of COP5 and COP7 may comprise
residues, e.g., 35 and 36, of an osteogenic protein embodying
invention.
[0020] Thus, in one aspect, the invention comprises a protein
comprising an amino acid sequence sufficiently duplicative of the
sequence of COP5 or COP7 such that it is capable of inducing
endochondral bone formation when properly folded and implanted in a
mammal in association with a matrix. Some of these sequences induce
cartilage, but not bone. Also, the bone forming materials may be
used to produce cartilage if implanted in an avascular locus, or if
an inhibitor to full bone development is implanted together with
the active protein. Thus, in another aspect, the invention
comprises a protein less than about 200 amino acids long in a
sequence sufficiently duplicative of the sequence of COP5 or COP7
such that it is capable at least of cartilage formation when
properly folded and implanted in a mammal in association with a
matrix.
[0021] In one preferred aspect, these proteins comprise species of
the generic amino acid sequences:
2 1 10 20 30 40 50 LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 60
70 80 90 100 QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX
or 1 10 20 30 40 50
CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 60 70 80 90 100
QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX
[0022] where the letters indicate the amino acid residues of
standard single letter code, and the Xs represent amino acid
residues. Preferred amino acid sequences within the foregoing
generic sequences are:
3 1 10 20 30 40 50 LYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV K
S S L QE VIS E FD Y E A AY MPESMKAS VI F E K I DN L N S Q ITK F P
TL A S K 60 70 80 90 100
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKHYQ- DMVVEGCGCR SI HAI SEQV
EP A EQMNSLAI FFNDQDK I RK EE T DA H H RF T S K DPV V Y N S H RN RS
N S K P E and 1 10 20 30 40 50
CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV RRRS K S S L QE
VIS E FD Y E A AY MPESMKAS VI KE F E K I DN L N S Q ITK F P TL Q A
S K 60 70 80 90 100 QTLVNSVNPGKIPKACCVPTELSAISML-
YLDENENVVLKNYQDMVVEGCGCR SI HAI SEQV EP A EQMNSLAI FFNDQDK I RK EE
T DA H H RF T S K DPV V Y N S H RN RS N S K P E
[0023] wherein each of the amino acids arranged vertically at each
position in the sequence may be used alternatively in various
combinations. Note that these generic sequences have 6 and
preferably 7 cysteine residues where inter- or intramolecular
disulfide bonds can form, and contain other critical amino acids
which influence the tertiary structure of the proteins. These
generic structural features are found in previously published
sequences, none of which have been described as capable of
osteogenic activity, and most of which never have been linked with
such activity.
[0024] Particular useful sequences include:
4 1 10 20 30 40 Vg1 CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE 50 60
70 ILNGSN--H-AILQTLVHSIEPED-IPLPCCVPTKM- SP 80 90 100
ISMLFYDNNDNVVLRHYENMAVDECGCR 1 10 20 30 40 DPP
CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPL- AD 50 60 70
HFNSTN--H-AVVQTLVNNNNPGK-VPKACCVPTQLDS 80 90 100
VAMLYLNDQSTVVLKNYQEMTVVGCGCR -5 HQRQA 1 10 20 30 40 OP1
CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA 80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH 1 10 20 30 40 CBMP-2a
CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 50 60 70
HLNSTN--H-AIVQTLVNSVNS-K-IPKACCVPTEL- SA 80 90 100
ISMLYLDENEKVVLKNYQDMVVEGCGCR 1 10 20 30 40 CBMP-2b
CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDC- PFPLAD 50 60 70
HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA 80 90 100
ISMLYLDEYDKVVLKNYQEMVVEGCGCR 1 10 20 30 40 CBMP-3
CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK 50 60 70
SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKM- SS 80 90 100
LSILFFDENKNVVLKVYPNMTVESCACR 1 10 20 30 40 COP1
LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFP- SAD 50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA 80 90 100
ISMLYLDENSTVVLKNYQEMTVVGCGCR 1 10 20 30 40 COP3
LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD 50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR 1 10 20 30 40 COP4
LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFP- SAD 50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR -10 PKHHSQRARKKNKN 1 10 20 30 40 COP16
CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD 50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
[0025] Vg1 is a known Xenopus sequence heretofore not associated
with bone formation. DPP is an amino acid sequence encoded by a
drosophila gene responsible for development of the dorsoventral
pattern. OP1 is a region of a natural sequence encoded by exons of
a genomic DNA sequence retrieved by applicants. The CBMPs are amino
acid sequences comprising subparts of mammalian proteins encoded by
genomic DNAs and cDNAs retrieved by applicants. The COPs are
biosynthetic protein sequences expressed by novel consensus gene
constructs, designed using the criteria set forth herein, and not
yet found in nature.
[0026] These proteins are believed to dimerize during refolding.
They appear not to be active when reduced. Various combinations of
species of the proteins, i.e., heterodimers, have activity, as do
homodimers. As far as applicants are aware, the COP5 and COP7
constructs constitute the first instances of the design of a
bioactive protein without preexisting knowledge of the active
region of a native form nucleotide or amino acid sequence.
[0027] The invention also provides native forms of osteogenic
protein, extracted from bone or produced using recombinant DNA
techniques. The substantially pure osteogenic protein may include
forms having varying glycosylation patterns, varying N-termini, a
family of related proteins having regions of amino acid sequence
homology, and active truncated or mutated forms of native protein,
no matter how derived. The osteogenic protein in its native form is
glycosylated and has an apparent molecular weight of about 30 kD as
determined by SDS-PAGE. When reduced, the 30 kD protein gives rise
to two glycosylated polypeptide chains having apparent molecular
weights of about 16 kD and 18 kD. In the reduced state, the 30 kD
protein has no detectable osteogenic activity. The deglycosylated
protein, which has osteogenic activity, has an apparent molecular
weight of about 27 kD. When reduced, the 27 kD protein gives rise
to the two deglycosylated polypeptides have molecular weights of
about 14 kD to 16 kD.
[0028] Analysis of digestion fragments indicate that the native 30
kD osteogenic protein contains the following amino acid sequences
(question marks indicate undetermined residues):
5 (1) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K; (2)
S-L-K-P-S-N-Y-A-T-I-Q-S-I-V; (3)
A-C-C-V-P-T-E-L-S-A-I-S-M-L-Y-L-D-E-N-E-K; (4)
M-S-S-L-S-I-L-F-F-D-E-N-K; (5) S-Q-E-L-V-D-F-Q-R; (6)
F-L-H-C-Q-F-S-E-R-N-S; (7) T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y; (8)
L-Y-D-P-M-V-V; (9) V-G-V-V-P-G-I-P-E-P-C-C-V-P-E; (10)
V-D-F-A-D-I-G; (11) V-P-K-P-C-C-A-P-T; (12) I-N-I-A-N-Y-L; (13)
D-N-H-V-L-T-M-F-P-I-A-I-N; (14) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-?-P;
(15) D-I-G-?-S-E-W-I-I-?-P; (16) S-I-V-R-A-V-G-V-P-G-I-P-E-P-?-?-V;
(17) D-?-I-V-A-P-P-Q-Y-H-A-F-Y; (18)
D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E; (19)
S-Q-T-L-Q-F-D-E-Q-T-L-K-?-A-R-?-K-Q; (20)
D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-E-P-R-N-?-A-R-R-Y-L; (21)
A-R-R-K-Q-W-I-E-P-R-N-?-A-?-R-Y-?-?-V-D; and (22)
R-?-Q-W-I-E-P-?-N-?-A-?-?-Y-L-K-V-D-?-A-?-?-G.
[0029] The substantially pure (i.e., free of contaminating proteins
having no osteoinductive activity) osteogenic proteins and the
synthetics are useful in clinical applications in conjunction with
a suitable delivery or support system (matrix). The matrix is made
up of particles or porous materials. The pores must be of a
dimension to permit progenitor cell migration and subsequent
differentiation and proliferation. The particle size should be
within the range of 70-850 mm, preferably 70-420 mm. It may be
fabricated by close packing particulate material into a shape
spanning the bone defect, or by otherwise structuring as desired a
material that is biocompatible (non-inflammatory) and,
biodegradable in vivo to serve as a "temporary scaffold" and
substratum for recruitment of migratory progenitor cells, and as a
base for their subsequent anchoring and proliferation. Currently
preferred carriers include particulate, demineralized, guanidine
extracted, species-specific (allogenic) bone, and particulate,
deglycosglated, protein extracted, demineralized, xenogenic bone.
Optionally, such xenogenic bone powder matrices also may be treated
with proteases such as trypsin. Other useful matrix materials
comprise collagen, homopolymers and copolymers of glycolic acid and
lactic acid, hydroxyapatite, tricalcium phosphate and other calcium
phosphates.
[0030] The availability of the protein in substantially pure form,
and knowledge of its amino acid sequence and other structural
features, enable the identification, cloning, and expression of
native genes which encode osteogenic proteins. When properly
modified after translation, incorporated in a suitable matrix, and
implanted as disclosed herein, these proteins are operative to
induce formation of cartilage and endochondral bone.
[0031] The consensus DNA sequences are also useful as probes for
extracting genes encoding osteogenic protein from genomic and cDNA
libraries. One of the consensus sequences has been used to isolate
a heretofore unidentified genomic DNA sequence, portions of which
when ligated encode a protein having a region capable of inducing
endochondral bone formation. This protein, designated OP1, has an
active region having the sequence set forth below.
6 1 10 20 30 40 OP1 LVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKP- CCAPTQLNA 80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH A longer active sequence is: -5 HQRQA
1 10 20 30 40 OP1 CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 50 60
70 YMNATN--H-AIVQTLVHFINPET-VPKPC- CAPTQLNA 80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH FIG. 1A discloses the genomic DNA
sequence of OP1.
[0032] The probes have also retrieved the DNA sequences identified
in PCT/087/01537, referenced above, designated therein as BMPII(b)
and BMPIII. The inventors herein have discovered that certain
subparts of these genomic DNAs, and BMPIIa, from the same
publication, when properly assembled, encode proteins (CBMPIIa,
CBMPIIb, and CBMPIII) which have true osteogenic activity, i.e.,
induce the full cascade of events when properly implanted in a
mammal leading to endochondral bone formation.
[0033] Thus, in view of this disclosure, skilled genetic engineers
can design and synthesize genes or isolate genes from cDNA or
genomic libraries which encode appropriate amino acid sequences,
and then can express them in various types of host cells, including
both procaryotes and eucaryotes, to produce large quantities of
active proteins in native forms, truncated analogs, muteins, fusion
proteins, and other constructs capable of inducing bone formation
in mammals including humans.
[0034] The osteogenic proteins and implantable osteogenic devices
enabled and disclosed herein will permit the physician to obtain
optimal predictable bone formation to correct, for example,
acquired and congenital craniofacial and other skeletal or dental
anomalies (Glowacki et al. (1981) Lancet 1:959-963). The devices
may be used to induce local endochondral bone formation in
non-union fractures as demonstrated in animal tests, and in other
clinical applications including periodontal applications where bone
formation is required. The other potential clinical application is
in cartilage repair, for example, in the treatment of
osteoarthritis.
BRIEF DESCRIPTION OF THE DRAWING
[0035] The foregoing and other objects of this invention, the
various features thereof, as well as the invention itself, may be
more fully understood from the following description, when read
together with the accompanying drawings, in which:
[0036] FIG. 1A represents the nucleotide sequence of the genomic
copy of osteogenic protein "OP1" gene. The unknown region between
1880 and 1920 actually represents about 1000 nucleotides;
[0037] FIG. 1B is a representation of the hybridization of the
consensus gene/probe to the osteogenic protein "OP1" gene;
[0038] FIG. 2 is a collection of plots of protein concentration (as
indicated by optical absorption) vs elution volume illustrating the
results of bovine osteogenic protein (BOP) fractionation during
purification on heparin-Sepharose-I; HAP-Ultragel; sieving gel
(Sephacryl 300); and heparin-Sepharose-II;
[0039] FIG. 3 is a photographic reproduction of a Coomassie blue
stained SDS polyacrylamide gel of the osteogenic protein under
non-reducing (A) and reducing (B) conditions;
[0040] FIG. 4 is a photographic reproduction of a Con A blot of an
SDS polyacrylamide gel showing the carbohydrate component of
oxidized (A) and reduced (B) 30 kD protein;
[0041] FIG. 5 is a photographic reproduction of an autoradiogram of
an SDS polyacrylamide gel of .sup.125I-labelled glycosylated (A)
and deglycosylated (B) osteogenic protein under non-reducing (1)
and reducing (2) conditions;
[0042] FIG. 6 is a photographic reproduction of an autoradiogram of
an SDS polyacrylamide gel of peptides produced upon the digestion
of the 30 kD osteogenic protein with V-8 protease (B), Endo Lys C
protease (C), pepsin (D), and trypsin (E). (A) is control;
[0043] FIG. 7 is a collection of HPLC chromatograms of tryptic
peptide digestions of 30 kD BOP (A), the 16 kD subunit (B), and the
18 kD subunit (C);
[0044] FIG. 8 is an HPLC chromatogram of an elution profile on
reverse phase C-18 HPLC of the samples recovered from the second
heparin-Sepharose chromatography step (see FIG. 2D). Superimposed
is the percent bone formation in each fraction;
[0045] FIG. 9 is a gel permeation chromatogram of an elution
profile on TSK 3000/2000 gel of the C-18 purified osteogenic peak
fraction. Superimposed is the percent bone formation in each
fraction;
[0046] FIG. 10 is a collection of graphs of protein concentration
(as indicated by optical absorption) vs. elution volume
illustrating the results of human protein fractionation on
heparin-Sepharose I (A), HAP-Ultragel (B), TSK 3000/2000 (C), and
heparin-Sepharose II (D). Arrows indicate buffer changes;
[0047] FIG. 11 is a graph showing representative dose response
curves for bone-inducing activity in samples from various
purification steps including reverse phase HPLC on C-18 (A),
Heparin-Sepharose II (B), TSK 3000 (C), HAP-ultragel (D), and
Heparin-Sepharose I (E);
[0048] FIG. 12 is a bar graph of radiomorphometric analyses of
feline bone defect repair after treatment with an osteogenic device
(A), carrier control (B), and demineralized bone (C);
[0049] FIG. 13 is a schematic representation of the DNA sequence
and corresponding amino acid sequence of a consensus gene/probe for
osteogenic protein (COPO);
[0050] FIG. 14 is a graph of osteogenic activity vs. increasing
molecular weight showing peak bone forming activity in the 30 kD
region of an SDS polyacrylamide gel;
[0051] FIG. 15 is a photographic representation of a Coomassie blue
stained SDS gel showing gel purified subunits of the 30 kD
protein;
[0052] FIG. 16 is a pair of HPLC chromatograms of Endo Asp N
proteinase digests of the 18 kD subunit (A) and the 16 kD subunit
(B);
[0053] FIG. 17 is a photographic representation of the histological
examination of bone implants in the rat model: carrier alone (A);
carrier and glycosylated osteogenic protein (B); and carrier and
deglycosylated osteogenic protein (C). Arrows indicate
osteoblasts;
[0054] FIG. 18 is a comparison of the amino acid sequence of
various osteogenic proteins to those of the TGF-beta family. COP1,
COP3, COP4, COP5, and COP7 are a family of analogs of synthetic
osteogenic proteins developed from the consensus gene that was
joined to a leader protein via a hinge region having the sequence
D-P-N-G that permitted chemical cleavage at the D-P site (by acid)
or N-G (by hydroxylamine) resulting in the release of the analog
protein; VGI is a Xenopus protein, DPP is a Drosophila protein; OP1
is a native osteogenic protein; CBMP2a and 2b, and CBMP3 are
subparts of proteins disclosed in PCT application 087/01537; MIS is
Mullerian inhibitory substance; and "consensus choices" represent
various substitutions of amino acids that may be made at various
positions in osteogenic proteins;
[0055] FIG. 19 is a graph illustrating the activity of xenogenic
matrix (deglycolylated bovine matrix);
[0056] FIGS. 20A and 20B are bar graphs showing the specific
activity of naturally sourced OP before and after gel elution as
measured by calcium content vs. increasing concentrations of
proteins (dose curve, in ng);
[0057] FIG. 21A is an E coli expression vector containing a gene of
an osteogenic protein fused to a leader protein;
[0058] FIG. 21B is the DNA sequence comprising a modified trp-LE
leader, two Fb domains of protein A, an ASP-PRO cleavage site, and
the COP5 sequence;
[0059] FIGS. 22A and 22B are photomicrographs of implants showing
the histology (day 12) of COP5 active recombinant protein. A is a
control (rat matrix alone, 25 mg). B is rat matrix plus COP5,
showing +++ cartilage formation and ++ bone formation (see key
infra). Similar results are achieved with COP7.
DESCRIPTION
[0060] Purification protocols have been developed which enable
isolation of the osteogenic protein present in crude protein
extracts from mammalian bone. While each of the separation steps
constitute known separation techniques, it has been discovered that
the combination of a sequence of separations exploiting the
protein's affinity for heparin and for hydroxyapatite (HAP) in the
presence of a denaturant such as urea is key to isolating the pure
protein from the crude extract. These critical separation steps are
combined with separations on hydrophobic media, gel exclusion
chromatography, and elution form SDS PAGE.
[0061] The isolation procedure enables the production of
significant quantities of substantially pure osteogenic protein
from any mammalian species, provided sufficient amounts of fresh
bone from the species is available. The empirical development of
the procedure, coupled with the availability of fresh calf bone,
has enabled isolation of substantially pure bovine osteogenic
protein (BOP). BOP has been characterized significantly as set
forth below; its ability to induce cartilage and ultimately
endochondral bone growth in cat, rabbit, and rat have been studied;
it has been shown to be able to induce the full developmental
cascade of bone formation previously ascribed to unknown protein or
proteins in heterogeneous bone extracts; and it may be used to
induce formation of endochondral bone in orthopedic defects
including non-union fractures. In its native form it is a
glycosylated, dimeric protein. However, it is active in
deglycosylated form. It has been partially sequenced. Its primary
structure includes the amino acid sequences set forth herein.
[0062] Elucidation of the amino acid sequence of BOP enables the
construction of pools of nucleic acid probes encoding peptide
fragments. Also, a consensus nucleic acid sequence designed as
disclosed herein based on the amino acid sequence data, inferred
codons for the sequences, and observation of partial homology with
known genes, also may be used as a probe. The probes may be used to
isolate naturally occuring cDNAs which encode active mammalian
osteogenic proteins (OP) as described below using standard
hybridization methodology. The mRNAs are present in the cytoplasm
of cells of various species which are known to synthesize
osteogenic proteins. Useful cells harboring the mRNAs include, for
example, osteoblasts from bone or osteosarcoma, hypertrophic
chondrocytes, and stem cells. The mRNAs can be used to produce cDNA
libraries. Alternatively, relevant DNAs encoding osteogenic protein
may be retrieved from cloned genomic DNA libraries from various
mammalian species.
[0063] The consensus sequence described above also may be refined
by comparison with the sequences present in certain regulatory
genes from drosophila, xenopus, and human followed by point
mutation, expression, and assay for activity. This approach has
been successful in producing several active totally synthetic
constructs not found in nature (as far as applicants are aware)
which have true osteogenic activity.
[0064] These discoveries enable the construction of DNAs encoding
totally novel, non-native protein constructs which individually,
and combined are capable of producing true endochondral bone. They
also permit expression of the natural material, truncated forms,
muteins, analogs, fusion proteins, and various other variants and
constructs, from cDNAs retrieved from natural sources or
synthesized using the techniques disclosed herein using automated,
commercially available equipment. The DNAs may be expressed using
well established recombinant DNA technologies in procaryotic or
eucaryotic host cells, and may be oxidized and refolded in vitro if
necessary for biological activity.
[0065] The isolation procedure for obtaining the protein from bone,
the retrieval of an osteogenic protein gene, the design and
production of biosynthetics, the nature of the matrix, and other
material aspects concerning the nature, utility, how to make, and
how to use the subject matter claimed herein will be further
understood from the following, which constitutes the best method
currently known for practicing the various aspects of the
invention.
I. Naturally Sourced Osteogenic Protein
[0066] A--Purification
[0067] A1. Preparation of Demineralized Bone
[0068] Demineralized bovine bone matrix is prepared by previously
published procedures (Sampath and Reddi (1983) Proc. Natl. Acad.
Sci. USA 80:6.591-6595). Bovine diaphyseal bones (age 1-10 days)
are obtained from a local slaughterhouse and used fresh. The bones
are stripped of muscle and fat, cleaned of periosteum, demarrowed
by pressure with cold water, dipped in cold absolute ethanol, and
stored at -20.degree. C. They are then dried and fragmented by
crushing and pulverized in a large mill. Care is taken to prevent
heating by using liquid nitrogen. The pulverized bone is milled to
a particle size between 70-420 mm and is defatted by two washes of
approximately two hours duration with three volumes of chloroform
and methanol (3:1). The particulate bone is then washed with one
volume of absolute ethanol and dried over one volume of anhydrous
ether. The defatted bone powder (the alternative method is to
obtain Bovine Cortical Bone Powder (75-425 mm) from American
Biomaterials) is then demineralized with 10 volumes of 0.5 N HCl at
4.degree. C. for 40 min., four times. Finally, neutralizing washes
are done on the demineralized bone powder with a large volume of
water.
[0069] A2. Dissociative Extraction and Ethanol Precipitation
[0070] Demineralized bone matrix thus prepared is dissociatively
extracted with 5 volumes of 4 M guanidine-HCl, 50 mM Tris-HCl, pH
7.0, containing protease inhibitors (5 mM benzamidine, 44 mM
6-aminohexanoic acid, 4.3 mM N-ethylmaleimide, 0.44 mm
phenylmethylsulfonyfluoride) for 16 hr. at 4.degree. C. The
suspension is filtered. The supernatant is collected and
concentrated to one volume using an ultrafiltration hollow fiber
membrane (Amicon, YM-10). The concentrate is centrifuged
(8,000.times.g for 10 min. at 4.degree. C.), and the supernatant is
then subjected to ethanol precipitation. To one volume of
concentrate is added five volumes of cold (-70.degree. C.) absolute
ethanol (100%), which is then kept at -70.degree. C. for 16 hrs.
The precipitate is obtained upon centrifugation at 10,000.times.g
for 10 min. at 4.degree. C. The resulting pellet is resuspended in
4 l of 85% cold ethanol incubated for 60 min. at -70.degree. C. and
recentrifuged. The precipitate is again resuspended in 85% cold
ethanol (2 l), incubated at -70.degree. C. for 60 min. and
centrifuged. The precipitate is then lyophilized.
[0071] A3. Heparin-Sepharose Chromatography I
[0072] The ethanol precipitated, lyophilized, extracted crude
protein is dissolved in 25 volumes of 6 M urea, 50 mM Tris-HCl, pH
7.0 (Buffer A) containing 0.15 M NaCl, and clarified by
centrifugation at 8,000.times.g for 10 min. The heparin-Sepharose
is column-equilibrated with Buffer A. The protein is loaded onto
the column and after washing with three column volume of initial
buffer (Buffer A containing 0.15 M NaCl), protein is eluted with
Buffer A containing 0.5 M NaCl. The absorption of the eluate is
monitored continuously at 280 nm. The pool of protein eluted by 0.5
M NaCl (approximately 1 column volumes) is collected and stored at
4.degree. C.
[0073] As shown in FIG. 2A, most of the protein (about 95%) remains
unbound. Approximately 5% of the protein is bound to the column.
The unbound fraction has no bone inductive activity when bioassayed
as a whole or after a partial purification through Sepharose
CL-6B.
[0074] A4. Hydroxyapaptite-Ultrogel Chromatography
[0075] The volume of protein eluted by Buffer A containing 0.5 M
NaCl from the heparin-Sepharose is applied directly to a column of
hydroxyapaptite-ultrogel (HAP-ultrogel) (LKB Instruments),
equilibrated with Buffer A containing 0.5 M NaCl. The HAP-ultrogel
is treated with Buffer A containing 500 mM Na phosphate prior to
equilibration. The unadsorbed protein is collected as an unbound
fraction, and the column is washed with three column volumes of
Buffer A containing 0.5 M NaCl. The column is subsequently eluted
with Buffer A containing 100 mM Na Phosphate (FIG. 2B).
[0076] The eluted component can induce endochondral bone as
measured by alkaline phosphatase activity and histology. As the
biologically active protein is bound to HAP in the presence of 6 M
urea and 0.5 M NaCl, it is likely that the protein has an affinity
for bone mineral and may be displaced only by phosphate ions.
[0077] A5. Sephacryl S-300 Gel Exclusion Chromatography
[0078] Sephacryl S-300 HR (High Resolution, 5 cm.times.100 cm
column) is obtained from Pharmacia and equilibrated with 4 M
guanidine-HCl, 50 mM Tris-HCl, pH 7.0. The bound protein fraction
from HA-ultrogel is concentrated and exhanged from urea to 4 M
guanidine-HCl, 50 mM Tris-HCl, pH 7.0 via an Amicon ultrafiltration
YM-10 membrane. The solution is then filtered with Schleicher and
Schuell CENTREX disposable microfilters. A sample aliquot of
approximately 15 ml containing approximately 400 mg of protein is
loaded onto the column and then eluted with 4 M guanidine-HCl, 50
mM Tris-HCl, pH 7.0, with a flow rate of 3 ml/min; 12 ml fractions
are collected over 8 hours and the concentration of protein is
measured at A.sub.280 nm (FIG. 2C). An aliquot of the individual
fractions is bioassayed for bone formation. Those fractions which
have shown bone formation and have a molecular weigh less than 35
kD are pooled and concentrated via an Amicon ultrafiltration system
with YM-10 membrane.
[0079] A6. Heparin-Sepharose Chromatography-II
[0080] The pooled osteo-inductive fractions obtained from gel
exclusion chromatography are dialysed extensively against distilled
water and then against 6 M urea, 50 mM Tris-HCl, pH 7.0 (Buffer A)
containing 0.1 M NaCl. The dialysate is then cleared through
centrifugation. The sample is applied to the heparin-sepharose
column (equilibrated with the same buffer). After washing with
three column volumes of initial buffer, the column is developed
sequentially with Buffer B containing 0.15 M NaCl, and 0.5 M NaCl
(FIG. 2D). The protein eluted by 0.5 M NaCl is collected and
dialyzed extensively against distilled water. It is then dialyzed
against 30% acetonitrile, 0.1% TFA at 4.degree. C.
[0081] A7. Reverse Phase HPLC
[0082] The protein is further purified by C-18 Vydac silica-based
HPLC column chromatography (particle size 5 mm; pore size 300 A).
The osteoinductive fraction obtained from heparin-sepharose-II
chromatograph is loaded onto the column, and washed in 0.1% TFA,
10% acetonitrile for five min. As shown in FIG. 8, the bound
proteins are eluted with a linear gradient of 10-30% acetonitrile
over 15 min., 30-50% acetonitrile over 60 min, and 50-70%
acetonitrile over 10 min at 22.degree. C. with a flow rate of 1.5
ml/min and 1.4 ml samples are collected in polycarbonate tubes.
Protein is monitored by absorbance at A.sub.214 nm. Column
fractions are tested for the presence of osteoinductive activity,
concanavalin A-blottable proteins and then pooled. Pools are then
characterized biochemically for the presence of 30 kD protein by
autoradiography, concanavalin A blotting, and Coomassie blue dye
staining. They are then assayed for in vivo osteogenic activity.
Biological activity is not found in the absence of 30 kD
protein.
[0083] A8. Gel Elution
[0084] The glycosylated or deglycosylated protein is eluted from
SDS gels (0.5 mm and 1.5 mm thickness) for further
characterization. .sup.125I-labelled 30 kD protein is routinely
added to each preparation to monitor yields. TABLE 1 shows the
various elution buffers that have been tested and the yields of
.sup.125I-labelled protein.
7TABLE 1 Elution of 30 kD Protein from SDS Gel % Eluted Buffer 0.5
mm 1.5 mm (1) dH.sub.2O 22 (2) 4M Guanidine-HCl, Tris-HCl, pH 7.0 2
(3) 4M Guanidine-HCl, Tris-HCl, pH 7.0, 93 52 0.5% Triton x 100 (4)
0.1% SDS, Tris-HCl, pH 7.0 98
[0085] TABLE 2 lists the steps used to isolate the 30 kD or
deglycosylated 27 kD gel-bound protein. The standard protocol uses
diffusion elution using 4M guanidine-HCl containing 0.5% Triton x
100 in Tris-HCl buffer or in Tris-HCl buffer containing 0.1% SDS to
achieve greater than 95% elution of the protein from the 27 or 30
kD region of the gel for demonstration of osteogenic activity in
vivo as described in later section.
[0086] In order to isolate substantially purified 30 kD or
deglycosylated 27 kD protein for sequencing and characterization,
the following steps are mentioned in Table 2.
8TABLE 2 Preparation of Gel Eluted Protein (C-18 Pool or
deglycoslated protein plus .sup.125I-labelled 30 kD protein) 1. Dry
using vacuum centrifugation; 2. Wash pellet with H.sub.2O; 3.
Dissolve pellet in gel sample buffer (no reducing agent); 4.
Electrophorese on pre-electrophoresed 0.5 mm mini gel; 5. Cut out
27 or 30 kD protein; 6. Elute from gel with 0.1% SDS, 50 mM
Tris-HCl, pH 7.0; 7. Filter through Centrex membrane; 8.
Concentrate in Centricon tube (10 kD membrane); 9. Chromatograph of
TSK-3000 gel filtration column; 10. Concentrate in Centricon
tube.
[0087] Chromatography in 0.1% SDS on a TSK-3000 gel filtration
column is performed to separate gel impurities, such as soluble
acrylamide, from the final product. The overall yield of labelled
30 kD protein from the gel elution protocol is 50-60% of the loaded
sample. Most of the loss occurs in the electrophoresis step, due to
protein aggregation and/or smearing. In a separate experiment, a
sample of gel eluted 30 kD protein is reduced, electrophoresed on
an SDS gel, and transferred to an Immobilon membrane. The membrane
is stained with Coomassie blue dye, cut into slices, and the slices
are counted. Coomassie blue dye stains the 16 kD and 18 kD reduced
species of the 30 kD protein almost exclusively. However, the
counts showed significant smearing throughout the gel in addition
to being concentrated in the 16 kD and 18 kD species. This suggests
that the .sup.125I-label can exhibit anomolous behavior on SDS gels
and cannot be used as an accurate marker for cold protein under
such circumstances.
[0088] The yield is 0.5 to 1.0 mg substantially pure osteogenic
protein per kg of bone.
[0089] A9. Isolation of the 16 kD and 18 kD Species
[0090] TABLE 3 summarizes the procedures involved in the
preparation of the subunits. Approximately 10 mg of gel eluted 30
kD protein (FIG. 3) is carboxymethylated and electrophoresed on an
SDS-gel. The sample contains .sup.125I-label to trace yields and to
use as an indicator for slicing the 16 kD, 18 kD and non-reduceable
30 K regions from the gel. FIG. 15 shows a Coomassie stained gel of
aliquots of the protein isolated from the different gel slices. The
slices corresponding to the 16 kD, 18 kD and non-reduceable 30 kD
species contained approximately 2-3 mg, 3-4 mg, and 1-2 mg, of
protein respectively, as estimated by staining intensity.
[0091] Prior to SDS electrophoresis, all of the 30 kD species can
be reduced to the 16 kD and 18 kD species. The nonreducible 30 kD
species observed after electrophoresis appears to be an artifact
resulting from the electrophoresis procedure.
9TABLE 3 Isolation of the Subunits of the 30 kD protein (C-18 pool
plus .sup.125I labeled 30 kD protein) 1. Electrophorese on SDS gel.
2. Cut out 30 kD protein. 3. Elute with 0.1% SDS, 50 nm Tris, pH
7.0. 4. Concentrate and wash with H.sub.2O in Centricon tube (10 kD
membranes). 5. Reduce and carboxymethylate in 1% SDS, 0.4 M Tris,
pH 8.5. 6. Concentrate and wash with H.sub.2O in Centricon tube. 7.
Electrophorese on SDS gel. 8. Cut out the 16 kD and 18 kD subunits.
9. Elute with 0.1% SDS, 50 mM Tris, pH 7.0. 10. Concentrate and
wash with H.sub.2O in Centricon tubes.
[0092] B. Demonstration that the 30 KD Protein is Osteogenic
Protein--Biological Characterization
[0093] B1. Gel Slicing:
[0094] Gel slicing experiments confirm that the isolated 30 kD
protein is the protein responsible for osteogenic activity.
[0095] Gels from the last step of the purification are sliced.
Protein in each fraction is extracted in 15 mM Tris-HCl, pH 7.0
containing 0.1% SDS or in buffer containing 4M guanidine-HCl, 0.5%
non-ionic detergent (Triton x 100), 50 mM Tris-HCl. The extracted
proteins are desalted, concentrated, and assayed for endochondral
bone formation activity. The results are set forth in FIG. 14. From
this Figure it is clear that the majority of osteogenic activity is
due to protein at 30 kD region of the gene. Activity in higher
molecular weight regions is apparently due to protein aggregation.
These protein aggregates, when reduced, yields the 16 kD and 18 kD
species discussed above.
[0096] B2. Con A-Sepharose Chromatography:
[0097] A sample containing the 30 kD protein is solubilized using
0.1% SDS, 50 mM Tris-HCl, and is applied to a column of Con
A-Sepharose equilibrated with the same buffer. The bound material
is eluted in SDS Tris-HCl buffer containing 0.5 M alpha-methyl
mannoside. After reverse phase chromatography of both the bound and
unbound fractions, Con A-bound materials, when implanted, result in
extensive bone formation. Further characterization of the bound
materials show a Con A-blottable 30 kD protein. Accordingly, the 30
kD glycosylated protein is responsible for the bone forming
activity.
[0098] B3. Gel Permeation Chromatography:
[0099] TSK-3000/2000 gel permeation chromatography in guanidine-HCl
alternately is used to achieve separation of the high specific
activity fraction obtained from C-18 chromatography (FIG. 9). The
results demonstrate that the peak of bone inducing activity elutes
in fractions containing substantially pure 30 kD protein by
Coomassie blue staining. When this fraction is iodinated and
subjected to autoradiography, a strong band at 30 kD accounts for
90% of the iodinated proteins. The fraction induces bone formation
in vivo at a dose of 50 to 100 ng per implant.
[0100] B4. Structural Requirements for Biological Activity
[0101] Although the role of 30 kD osteogenic protein is clearly
established for bone induction, through analysis of proteolytic
cleavage products we have begun to search for a minimum structure
that is necessary for activity in vivo. The results of cleavage
experiments demonstrate that pepsin treatment fails to destroy bone
inducing capacity, whereas trypsin or CNBr completely abolishes the
activity.
[0102] An experiment is performed to isolate and identify pepsin
digested product responsible for biological activity. Sample used
for pepsin digest were 20%-30% pure. The buffer used is 0.1% TFA in
water. The enzyme to substrate ratio is 1:10. A control sample is
made without enzyme. The digestion mixture is incubated at room
temperature for 16 hr. The digested product is then separated in 4
M guanidine-HCl using gel permeation chromatography, and the
fractions are prepared for in vivo assay. The results demonstrate
that active fractions from gel permeation chromatography of the
pepsin digest correspond to molecular weight of 8 kD-10 kD.
[0103] In order to understand the importance of the carbohydrates
moiety with respect to osteogenic activity, the 30 kD protein has
been chemically deglycosylated using HF (see below). After
analyzing an aliquot of the reaction product by Con A blot to
confirm the absence of carbohydrate, the material is assayed for
its activity in vivo. The bioassay is positive (i.e., the
deglycosylated protein produces a bone formation response as
determined by histological examination shown in FIG. 17C),
demonstrating that exposure to HF did not destroy the biological
function of the protein. In addition, the specific activity of the
deglycosylated protein is approximately the same as that of the
native glycosylated protein.
[0104] B5. Specific Activity of BOP
[0105] Experiments were performed 1) to determine the half maximal
bone-inducing activity based on calcium content of the implant; 2)
to estimate proteins at nanogram levels using a gel scanning
method; and 3) to establish dose for half maximal bone inducing
activity for gel eluted 30 kD BOP. The results demonstrate that gel
eluted substantially pure 30 kD osteogenic protein induces bone at
less than 5 ng per 25 mg implant and exhibits half maximal bone
differentiation activity at 20 ng per implant. The purification
data suggest that osteogenic protein has been purified from bovine
bone to 367,307 fold after final gel elution step with a specific
activity of 47,750 bone forming units per mg of protein.
[0106] B5(a)Half Maximal Bone Differentiation Activity
[0107] The bone inducing activity is determined biochemically by
the specific activity of alkaline phosphatase and calcium content
of the day 12 implant. An increase in the specific activity of
alkaline phosphatase indicates the onset of bone formation. Calcium
content, on the other hand, is proportional to the amount of bone
formed in the implant. The bone formation is therefore calculated
by determining calcium content of the implant on day 12 in rats and
expressed as bone forming units, which represent the amount that
exhibits half maximal bone inducing activity compared to rat
demineralized bone matrix. Bone induction exhibited by intact
demineralized rat bone matrix is considered to be the maximal
bone-differentiation activity for comparison.
[0108] B5(b)Protein Estimation Using Gel Scanning Techniques
[0109] A standard curve is developed employing known amounts of a
standard protein, bovine serum albumin. The protein at varying
concentration (50-300 ng) is loaded on 15% SDS gel,
electrophoresed, stained in comassie and destained. The gel
containing standard proteins is scanned at predetermined settings
using a gel scanner at 580 nm. The area covered by the protein band
is calculated and a standard curve against concentrations of
protein is constructed. A sample with an unknown protein
concentration is electrophoresed with known concentration of BSA.
The lane contained unknown sample is scanned and from the area the
concentration of Protein is determined.
[0110] B5(c)Gel Elution and Specific Activity
[0111] An aliquot of C-18 highly purified active fraction is
subjected to SDS gel and sliced according to molecular weights
described in FIG. 14. Proteins are eluted from the slices in 4 M
guanidine-HCl containing 0.5% Triton X-100, desalted, concentrated
and assayed for endochondral bone forming activity as determined by
calcium content. The C-18 highly active fractions and gel eluted
substantially pure 30 kD osteogenic protein are implanted in
varying concentrations in order to determine the half maximal bone
inducing activity.
[0112] FIG. 14 demonstrates that the bone inducing activity is due
to proteins eluted at 28-34 kD region. The recovery of activity
after gel elution step is determined by calcium content. FIGS. 20A
and 20B represent the bone inducing activity for the various
concentrations of 30 kD protein before and after gel elution as
estimated by calcium content. The concentration of protein is
determined by gel scanning in the 30 kD region. The data suggest
that the half maximal activity for 30 kD protein before gel elution
is 69 nanogram per 25 mg implant and is 21 nanogram per 25 mg
implant after elution. Table 4 describes the yield, total specific
activity, and fold purification of osteogenic protein at each step
during purification. Approximately 500 ug of heparin sepharose I
fraction, 130-150 ug of the HA ultrogel fraction, 10-12 ug of the
gel filtration fraction, 4-5 ug of the heparin sepharose II
fraction, 0.4-0.5 ug of the C-18 highly purified fraction, and
20-25 ng of gel eluted substantially purified is needed per 25 mg
of implant for unequivocal bone formation for half maximal
activity. Thus, 0.8-1.0 ng purified osteogenic protein per mg of
implant is required to exhibit half maximal bone differentiation
activity in vivo.
10TABLE 4 PURIFICATION OF BOP Biological Specific Purification
Protein Activity Activity Purification Steps (mg.) Units* Units/mg.
Fold Ethanol 30,000# 4,000 0.13 1 Precipitate** Heparin 1,200#
2,400 2.00 15 Sepharose I HA-Ultrogel 300# 2,307 7.69 59 Gel
filtration 20# 1,600 80.00 615 Heparin 5# 1,000 200.00 1,538
Sepharose II C-18 HPLC 0.070@ 150 2,043.00 15,715 Gel elution
0.004@ 191 47,750.00 367,307 Values are calculated from 4 kg. of
bovine bone matrix (800 g of demineralized matrix). *One unit of
bone forming activity is defined as the amount that exhibits half
maximal bone differentiation activity compared to rat demineralized
bone matrix, as determined by calcium content of the implant on day
12 in rats. #Proteins were measured by absorbance at 280 nm.
@Proteins were measured by gel scanning method compared to known
standard protein, bovine serum albumin. **Ethanol-precipitated
guanidine extract of bovine bone is a weak inducer of bone in rats,
possibly due to endogenous inhibitors. This precipitate is
subjected to gel filtration and proteins less than 50 kD were
separated and used for bioassay.
[0113] C. Chemical Characterization of BOP
[0114] C1. Molecular Weight and Structure
[0115] Electrophoresis of the most active fractions from reverse
phase C-18 chromatography on non-reducing SDS polyacrylamide gels
reveals a single band at about 30 kD as detected by both Coomassie
blue staining (FIG. 3A) and autoradiography.
[0116] In order to extend the analysis of BOP, the protein was
examined under reducing conditions. FIG. 3B shows an SDS gel of BOP
in the presence of dithiothreitol. Upon reduction, 30 kD BOP yields
two species which are stained with Coomassic blue dye: a 16 kD
species and an 18 kD species. Reduction causes loss of biological
activity. Methods for the efficient elution of the proteins from
SDS gels have been tested, and a protocol has been developed to
achieve purification of both proteins. The two reduced BOP species
have been analyzed to determine if they are structurally related.
Comparison of the amino acid composition of the two proteins (as
disclosed below) shows little differences, indicating that the
native protein may comprise, two chains having some homology.
[0117] C2. Charge Determination
[0118] Isoelectric focusing studies are initiated to further
evaluate the 30 kD protein for possibile heterogeneity. Results to
date have not revealed any such heterogeneity. The oxidized and
reduced species migrate as diffuse bands in the basic region of the
isoelectric focusing gel, using the iodinated 30 kD protein for
detection. Using two dimensional gel electrophoresis and Con A for
detection, the oxidized 30 kD protein show one species migrating in
the same basic region as the iodinated 30 kD protein. The diffuse
character of the band may be traced to the presence of carbohydrate
attached to the protein.
[0119] C3. Presence of Carbohydrate
[0120] The 30 kD protein has been tested for the presence of
carbohydrate by Concanavalin A (Con A) blotting after SDS-PAGE and
transfer to nitrocellulose paper. The results demonstrate that the
30 kD protein has a high affinity for Con A, indicating that the
protein is glycosylated (FIG. 4A). In addition, the Con A blots
provide evidence for a substructure in the 30 kD region of the gel,
suggesting heterogeneity due to varying degrees of glycosylation.
After reduction (FIG. 4B), Con A blots show evidence for two major
components at 16 kD and 18 kD. In addition, it has been
demonstrated that no glycosylated material remains at the 30 kD
region after reduction.
[0121] In order to confirm the presence of carbohydrate and to
estimate the amount of carbohydrate attached, the 30 kD protein is
treated with N-glycanase, a deglycosylating enzyme with a broad
specificity. Samples of the .sup.125I-labelled 30 kD protein are
incubated with the enzyme in the presence of SDS for 24 hours at
37.degree. C. As observed by SDS-PAGE, the treated samples appear
as a prominent species at about 27 kD (FIG. 5A). Upon reduction,
the 27 kD species is reduced to species having a molecular weight
of about 14 kD-16 kD (FIG. 5B).
[0122] Chemical cleavage of the carbohydrate moieties using
hydrogen fluoride (HF) is performed to assess the role of
carbohydrate on the bone inducing activity of BOP in vivo. Active
osteogenic protein fractions pooled from the C-18 chromatography
step are dried in vacuo over P.sub.2O.sub.5 in a polypropylene
tube, and 50 ml freshly distilled anhydrous HF at -70.degree. C. is
added. After capping the tube tightly, the mixture is kept at
0.degree. C. in an ice-bath with occasional agitation for 1 hr. The
HF is then evaporated using a continuous stream of dry nitrogen
gas. The tube is removed from the ice bath and the residue dried in
vacuo over P.sub.2O.sub.5 and KOH pellets.
[0123] Following drying, the samples are dissolved in 100 ml of 50%
acetonitrile/0.1% TFA and aliquoted for SDS gel analysis, Con A
binding, and biological assay. Aliquots are dried and dissolved in
either SDS gel sample buffer in preparation for SDS gel analysis
and Con A blotting or 4 M guanidine-HCl, 50 mM Tris-HCl, pH 7.0 for
biological assay.
[0124] The results show that samples are completely deglycosylated
by the HF treatment: Con A blots after SDS gel electrophoreses and
transfer to Immobilon membrane showed no binding of Con A to the
treated samples, while untreated controls were strongly positive at
30 kD. Coomassie gels of treated samples showed the presense of a
27 kD band instead of the 30 kD band present in the untreated
controls.
[0125] C4. Chemical and Enzymatic Cleavage
[0126] Cleavage reactions with CNBr are analyzed using Con A
binding for detection of fragments associated with carbohydrate.
Cleavage reactions are conducted using trifluoroacetic acid (TFA)
in the presence and absence of CNBr. Reactions are conducted at
37.degree. C. for 11 hours, and the samples are vacuum dried. The
samples are washed with water, dissolved in SDS gel sample buffer
with reducing agent, boiled and applied to an SDS gel. After
electrophoresis, the protein is transferred to Immobilon membrane
and visualized by Con A binding. In low concentrations of acid
(1%), CNBr cleaves the majority of 16 kD and 18 kD species to one
product, a species about 14 kD. In reactions using 10% TFA, a 14 kD
species is observed both with and without CNBr.
[0127] Four proteolytic enzymes are used in these experiments to
examine the digestion products of the 30 kD protein: 1) V-8
protease; 2) Endo Lys C protease; 3) pepsin; and 4) trypsin. Except
for pepsin, the digestion buffer for the enzymes is 0.1 M ammonium
bicarbonate, pH 8.3. The pepsin reactions are done in 0.1% TFA. The
digestion volume is 100 ml and the ratio of enzyme to substrate is
1:10. .sup.125I-labelled 30 kD osteogenic protein is added for
detection. After incubation at 37.degree. C. for 16 hr., digestion
mixtures are dried down and taken up in gel sample buffer
containing dithiothreitol for SDS-PAGE. FIG. 6 shows an
autoradiograph of an SDS gel of the digestion products. The results
show that under these conditions, only trypsin digests the reduced
16 kD/18 kD species completely and yields a major species at around
12 kD. Pepsin digestion yields better defined, lower molecular
weight species. However, the 16 kD/18 kD fragments were not
digested completely. The V-8 digest shows limited digestion with
one dominant species at 16 kD.
[0128] C5. Protein Sequencing
[0129] To obtain amino acid sequence data, the protein is cleaved
with trypsin or Endoproteinase Asp-N (EndoAsp-N). The tryptic
digest of reduced and carboxymethylated 30 kD protein
(approximately 10 mg) is fractionated by reverse-phase HPLC using a
C-8 narrowbore column (13 cm.times.2.1 mm ID) with a
TFA/acetonitrile gradient and a flow rate of 150 ml/min. The
gradient employs (A) 0.06% TFA in water and (B) 0.04% TFA in water
and acetonitrile (1:4; v:v). The procedure was 10% B for five min.,
followed by a linear gradient for 70 min. to 80% B, followed by a
linear gradient for 10 min. to 100% B. Fractions containing
fragments as determined from the peaks in the HPLC profile (FIG.
7A) are rechromatographed at least once under the same conditions
in order to isolate single components satisfactory for sequence
analysis.
[0130] The HPLC profiles of the similarly digested 16 kD and 18 kD
subunits are shown in FIGS. 7B and 7C, respectively. These peptide
maps are similar suggesting that the subunits are identical or are
closely related.
[0131] The 16 kD and 18 kD subunits are digested with Endo Asp N
proteinase. The protein is treated with 0.5 mg EndoAsp-N in 50 mM
sodium phosphate buffer, pH 7.8 at 36.degree. C. for 20 hr. The
conditions for fractionation are the same as those described
previously for the 30 kD, 16 kD, and 18 kD digests. The profiles
obtained are shown in FIGS. 16A and 16B.
[0132] Various of the peptide fragments produced using the
foregoing procedures have been analyzed in an automated amino acid
sequencer (Applied Biosystems 470A with 120A on-line PTH analysis).
The following sequence data has been obtained:
11 (1) S-F-D-A-Y-Y-C-S-G-A-C-Q-F-P-M-P-K; (2)
S-L-K-P-S-N-Y-A-T-I-Q-S-I-V; (3) A-C-C-V-P-T-E-L-S-A-I-S-
-M-L-Y-L-D-E-N-E-K; (4) M-S-S-L-S-I-L-F-F-D-E-N-K; (5)
S-Q-E-L-Y-V-D-F-Q-R; (6) F-L-H-C-Q-F-S-E-R-N-S; (7)
T-V-G-Q-L-N-E-Q-S-S-E-P-N-I-Y- ; (8) L-Y-D-P-M-V-V; (9)
V-G-V-V-P-G-I-P-E-P-C-C-V-P-E; (10) V-D-F-A-D-I-G; (11)
V-P-K-P-C-C-A-P-T; (12) I-N-I-A-N-Y-L; (13)
D-N-H-V-L-T-M-F-P-I-A-I-N; (14) D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-?-P;
(15) D-I-G-?-S-E-W-I-I-?-P; (16) S-I-V-R-A-V-G-V-P-G-I-P-E-P-?-
-?-V; (17) D-?-I-V-A-P-P-Q-Y-H-A-F-Y; (18)
D-E-N-K-N-V-V-L-K-V-Y-P-N-M-T-V-E; (19)
S-Q-T-L-Q-F-D-E-Q-T-L-K-?-A-R-?-K-Q; (20)
D-E-Q-T-L-K-K-A-R-R-K-Q-W-I-E-P-R-N-?-A-R-R-Y- L; (21)
A-R-R-K-Q-W-I-E-P-R-N-?-A-?-R-Y-?-?-V-D; and (22)
R-?-Q-W-I-E-P-?-N-?-A-?-?-Y-L-K-V-D-?-A-?-?-G
[0133] C6. Amino Acid Analysis
[0134] Strategies for obtaining amino acid composition were
developed using gel elution from 15% SDS gels, transfer onto
Immobilon, and hydrolysis. Immobilon membrane is a polymer of
vinylidene difluoride and, therefore, is not susceptible to acid
cleavage. Samples of oxidized (30 kD) and reduced (16 kD and 18 kD)
BOP are electrophoresed on a gel and transferred to Immobilon for
hydrolysis and analysis as described below. The composition data
generated by amino acid analyses of 30 kD BOP is reproducible, with
some variation in the number of residues for a few amino acids,
especially cysteine and isoleucine.
[0135] Samples are run on 15% SDS gels, transferred to Immobilon,
and stained with Coomassie blue. The bands of interest are excised
from the Immobilon, with a razor blade and placed in a 6.times.50
mm Corning test tube cleaned by pyrolysis at 550.degree. C. When
cysteine is to be determined, the samples are treated with
performic acid, which converts cysteine to cysteic acid. Cysteic
acid is stable during hydrolysis with HCl, and can be detected
during the HPLC analysis by using a modification of the normal
Pico-Tag eluents (Millipore) and gradient. The performic acid is
made by mixing 50 ml 30% hydrogen peroxide with 950 ml 99% formic
acid, and allowing this solution to stand at room temperature for 2
hr. The samples are then treated with performic acid (PFA); 20 ml
PFA is pippetted onto each sample and placed in an ice bath at
4.degree. C. for 2.5 hours. After 2.5 hr. the PFA is removed by
drying in vacuo, and the samples are then hydrolyzed. A standard
protein of known composition and concentration containing cysteine
is treated with PFA and hydrolyzed concurrently with the osteogenic
protein samples, to take as a control for hydrolysis and amino acid
chromatography.
[0136] The hydrolysis of the osteogenic protein samples is done in
vacuo. The samples, with empty tubes and Immobilon blanks, are
placed in a hydrolysis vessel which is placed in a dry ice/ethanol
bath to keep the HCl from prematurely evaporating. 200 ml 6 N HCl
containing 2% phenol and 0.1% stannous chloride are added to the
hydrolysis vessel outside the tubes containing the samples. The
hydrolysis vessel is then sealed, flushed with prepurified
nitrogen, evacuated, and then held at 115.degree. C. for 24 hours,
after which time the HCl is removed by drying in vacuo.
[0137] After hydrolysis, each piece of Immobilon is transferred to
a fresh tube, where it is rinsed twice with 100 ml 0.1% TFA, 50%
acetonitrile. The washings are returned to the original sample
tube, which is then redried as below. A similar treatment of amino
acid analysis on Immobilon can be found in the literature (LeGendre
and Matsudaira (1988) Biotechniques 6:154-159).
[0138] The samples are redried twice using 2:2:1
ethanol:water:triethylami- ne and allowed to dry at least 30 min.
after each addition of redry reagent. These redrying steps bring
the sample to the proper pH for derivatization.
[0139] The samples are derivatized using standard methodology. The
solution is added to each sample tube. The tubes are placed in a
desiccator which is partially evacuated, and are allowed to stand
for 20 min. The desiccator is then fully evacuated, and the samples
are dried for at least 3 hr. After this step the samples may be
stored under vacuum at -20.degree. C. or immediately diluted for
HPLC. The samples are diluted with Pico-Tag Sample Diluent
(generally 100 ml) and allowed to stand for 20 min., after which
they are analyzed on HPLC using the Pico Tag chromatographic system
with some minor changes involving gradients, eluents, initial
buffer conditions and oven temperature.
[0140] After HPLC analysis, the compositions are calculated. The
molecular weights are assumed to be 14.4 kD, 16.2 kD, and 27 kD to
allow for 10% carbohydrate content. The number of residues is
approximated by dividing the molecular weight by the average
molecular weight per amino acid, which is 115. The total picomoles
of amino acid recovered is divided by the number of residues, and
then the picomoles recovered for each amino acid is divided by the
number of picomoles per residue, determined above. This gives an
approximate theoretical number of residues of each amino acid in
the protein. Glycine content may be overestimated in this type of
analysis.
[0141] Composition data obtained are shown in TABLE 5.
12TABLE 5 BOP Amino Acid Analyses Amino Acid 30 kD 16 kD 18 kD
Aspartic Acid/ 22 14 15 Asparagine Glutamic Acid/ 24 14 16
Glutamine Serine 24 16 23 Glycine 29 18 26 Histidine 5 * 4 Arginine
13 6 6 Threonine 11 6 7 Alanine 18 11 12 Proline 14 6 6 Tyrosine 11
3 3 Valine 14 8 7 Methionine 3 0 2 Cysteine** 16 14 12 Isoleucine
15 14 10 Leucine 15 8 9 Phenylalanine 7 4 4 Tryptophan ND ND ND
Lysine 12 6 6 * This result is not integrated because histidine is
present in low quantities. **Cysteine is corrected by percent
normally recovered from performic acid hydrolysis of the standard
protein.
[0142] The results obtained from the 16 kD and 18 kD subunits, when
combined, closely resemble the numbers obtained from the native 30
kD protein. The high figures obtained for glycine and serine are
most likely the result of gel elution.
[0143] D. Purification of Human Osteogenic Protein
[0144] Human bone is obtained from the Bone Bank, (Mass. General
Hospital, Boston, Mass.), and is milled, defatted, demarrowed and
demineralized by the procedure disclosed above. 320 g of
mineralized bone matrix yields 70-80 g of demineralized bone
matrix. Dissociative extraction and ethanol precipitation of the
matrix gives 12.5 g of guanidine-HCl extract.
[0145] One third of the ethanol precipitate (0.5 g) is used for gel
filtration through 4 M guanidine-HCl (FIG. 10A). Approximately
70-80 g of ethanol precipitate per run is used. In vivo bone
inducing activity is localized in the fractions containing proteins
in the 30 kD range. They are pooled and equilibrated in 6 M urea,
0.5 M NaCl buffer, and applied directly onto a HAP column; the
bound protein is eluted stepwise by using the same buffer
containing 100 mM and 500 mM phosphate (FIG. 10B). Bioassay of HAP
bound and unbound fractions demonstrates that only the fraction
eluted by 100 mM phosphate has bone inducing activity in vivo. The
biologically active fraction obtained from HAP chromatography is
subjected to heparin-Sepharose affinity chromatography in buffer
containing low salt; the bound proteins are eluted by 0.5 M NaCl
(FIG. 10C). Assaying the heparin-Sepharose fractions shows that the
bound fraction eluted by 0.5M NaCl have bone-inducing activity. The
active fraction is then subjected to C-18 reverse phase
chromatography. (FIG. 10D).
[0146] The active fraction can then be subjected to SDS-PAGE as
noted above to yield a band at about 30 kD comprising substantially
pure human osteogenic protein.
E. Biosynthetic Probes for Isolation of Genes Encoding Native
Osteogenic Protein
[0147] E-1 PROBE DESIGN
[0148] A synthetic consensus gene shown in FIG. 13 was designed as
a hybridization probe (and to encode a consensus protein, see
below) based on amino acid predictions from homology with the
TGF-beta gene family and using human codon bias as found in human
TGF-beta. The designed concensus sequence was then constructed
using known techniques involving assembly of oligonucleotides
manufactured in a DNA synthesizer.
[0149] Tryptic peptides derived from BOP and sequenced by Edman
degradation provided amino acid sequences that showed strong
homology with the Drosophila DPP protein sequence (as inferred from
the gene), the Xenopus VG1 protein, and somewhat less homology to
inhibin and TGF-beta, as demonstrated below in TABLE 6.
13TABLE 6 protein amino acid sequence homology (BOP)
SFDAYYCSGACQFPS ***** * * ** (9/15 matches) (DPP) GYDAYYCHGKCPFFL
(BOP) SFDAYYCSGACQFPS * ** * * * (6/15 matches) (Vg1)
GYMANYCYGECPYPL (BOP) SFDAYYCSGACQFPS * ** * * (5/15 matches)
(inhibin) GYHANYCEGECPSHI (BOP) SFDAYYCSGACQFPS * * * * (4/15
matches) (TGF-beta) GYHANFCLGPCPYIW (BOP) K/RACCVPTELSAISMLYLDEN
***** * **** * * (12/20 matches) (Vg1) LPCCVPTKMSPISMLFYDNN (BOP)
K/RACCVPTELSAISMLYLDEN * ***** * **** * (12/20 matches) (inhibin)
KSCCVPTKLRPMSMLYYDDG (BOP) K/RACCVPTELSAISMLYLDE ***** * * (6/19
matches) (TGF-beta) APCCVPQALEPLPIVYYVG (BOP)
K/RACCVPTELSAISMLYLDEN ******* * **** (12/20 matches) (DPP)
KACCVPTQLDSVAMLAYLNDQ (BOP) LYVDF ***** (5/5 matches) (DPP) LYVDF
(BOP) LYVDF *** * (4/5 matches) (Vg1) LYVEF (BOP) LYVDF ** ** (4/5
matches) (TGF-beta) LYIDF (BOP) LYVDF * * (2/4 matches) (inhibin)
FFVSF * -match
[0150] In determining the amino acid sequence of an osteogenic
protein (from which the nucleic acid sequence can be determined),
the following points were considered: (1) the amino acid sequence
determined by Edman degradation of osteogenic protein tryptic
fragments is ranked highest as long as it has a strong signal and
shows homology or conservative changes when aligned with the other
members of the gene family; (2) where the sequence matches for all
four proteins, it is used in the synthetic gene sequence; (3)
matching amino acids in DPP and Vg1 are used; (4) If Vg1 or DPP
diverged but either one were matched by inhibin or by TGF-beta,
this matched amino acid is chosen; (5) where all sequences
diverged, the DPP sequence is initially chosen, with a later plan
of creating the Vg1 sequence by mutagenesis kept as a possibility.
In addition, the consensus sequence is designed to preserve the
disulfide crosslinking and the apparent structural homology.
[0151] One purpose of the originally designed synthetic consensus
gene sequence, designated COPO, (see FIG. 13), was to serve as a
probe to isolate natural genes. For this reason the DNA was
designed using human codon bias. Alternatively, probes may be
constructed using conventional techniques comprising a group of
sequences of nucleotides which encode any portion of the amino acid
sequence of the osteogenic protein produced in accordance with the
foregoing isolation procedure. Use of such pools of probes also
will enable isolation of a DNA encoding the intact protein.
[0152] E-2 Retrieval of Genes Encoding Osteogenic Protein from
Genomic Library
[0153] A human genomic library (Maniatis-library) carried in lambda
phage (Charon 4A) was screened using the COPO consensus gene as
probe. The initial screening was of 500,000 plaques (10 plates of
50,000 each). Areas giving hybridization signal were punched out
from the plates, phage particles were eluted and plated again at a
density of 2000-3000 plaques per plate. A second hybridization
yielded plaques which were plated once more, this time at a density
of ca 100 plaques per plate allowing isolation of pure clones. The
probe (COPO) is a 300 base pair BamHI-PstI fragment restricted from
an amplification plasmid which was labeled using alpha 32 dCTP
according to the random priming method of Feinberg and Vogelstein,
Anal. Biochem., 137, 266-267, 1984. Prehybridization was done for 1
hr in 5.times.SSPE, 10.times. Denhardt's mix, 0.5% SDS at
50.degree. C. Hybridization was overnight in the same solution as
above plus probe. The washing of nitrocellulose membranes was done,
once cold for 5 min. in 1.times.SSPE with 0.1% SDS and twice at
5.degree. C. for 2.times.30 min. in the same solution. Using this
procedure, twenty-four positive clones were found. Two of these
yielded the genes corresponding to BMP-2b, one yielded BMP-3 (see
PCT US 87/01537) and two contained a gene never before reported
designated OP1, osteogenic protein-1 described below.
[0154] Southern blot analysis of lambda #13 DNA showed that an
approximately 3 kb BamHI fragment hybridized to the probe. (See
FIG. 1B). This fragment was isolated and subcloned into a
bluescript vector (at the BamHI site). The clone was further
analyzed by Southern blotting and hybridization to the COPO probe.
This showed that a 1 kb (approx.) EcoRI fragment strongly
hybridized to the probe. This fragment was subcloned into the EcoRI
site of a bluescript vector, and sequenced. Analysis of this
sequence showed that the fragment encoded the carboxy terminus of a
protein, named osteogenic protein-1 (OP1). The protein was
identified by amino acid homology with the TGF-beta family. For
this comparison cysteine patterns were used and then the adjacent
amino acids were compared. Consensus splice signals were found
where amino acid homologies ended, designating exon intron
boundaries. Three exons were combined to obtain a functional
TGF-beta-like domain containing seven cysteines. Two introns were
deleted by looping out via primers bridging the exons using the
single stranded mutagenesis method of Kunkel. Also, upstream of the
first cysteine, an EcoRI site and an asp-pro junction for acid
cleavage were introduced, and at the 3' end a PstI site was added
by the same technique. Further sequence information (penultimate
exon) was obtained by sequencing the entire insert. The sequencing
was done by generating a set of unidirectionally deleted clones
(Ozkaynak, E., and Putney, S.: Biotechniques, 5, 770-773, 1987).
The obtained sequence covers about 80% of the TGF-beta-like region
of OP1 and is set forth in FIG. 1A. The complete sequence of the
TGF-beta like region was obtained by first subcloning all EcoRI
generated fragments of lambda clone #13 DNA and sequencing a 4 kb
fragment that includes the first portion of the TGF-beta like
region (third exon counting from end) as well as sequences
characterized earlier. The gene on an EcoRI to PstI fragment was
inserted into an E. coli expression vector controlled by the trp
promoter-operator to produce a modified trp LE fusion protein with
an acid cleavage site. The OP1 gene encodes amino acids
corresponding substantially to a peptide found in sequences of
naturally sourced material. The amino acid sequence of what is
believed to be its active region is set forth below:
14 1 10 20 30 40 OP1 LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 50 60 70
YMNATN--H-AIVQTLVHFINPET-VPK- PCCAPTQLNA 80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH
[0155] A longer active sequence is:
15 -5 HQRQA 1 10 20 30 40 OP1 CKKHELYVSFR-DLGWQDWIIAPEGY-
AAYYCEGECAFPLNS 50 60 70 YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA 80
90 100 ISVLYFDDSSNVILKKYRNMVVRACGCH
[0156] E-3 Probing cDNA Library
[0157] Another example of the use of pools of probes to enable
isolation of a DNA encoding the intact protein is shown by the
following. Cells known to express the protein are extracted to
isolate total cytoplasmic RNA. An oligo-dT column can be used to
isolate mRNA. This mRNA can be size fractionated by, for example,
gel electrophoresis. The fraction which includes the mRNA of
interest may be determined by inducing transient expression in a
suitable host cell and testing for the presence of osteogenic
protein using, for example, antibody raised against peptides
derived from the tryptic fragments of osteogenic protein in an
immunoassay. The mRNA fraction is then reverse transcribed to
single stranded cDNA using reverse transcriptase; a second
complementary DNA strand can then be synthesized using the cDNA as
a template. The double-standard DNA is then ligated into vectors
which are used to transfect bacteria to produce a cDNA library.
[0158] The radiolabelled consensus sequence, portions thereof,
and/or synthetic deoxy oligonucleotides complementary to codons for
the known amino acid sequences in the osteogenic protein may be
used to identify which of the DNAs in the cDNA library encode the
full length osteogenic protein by standard DNA-DNA hybridization
techniques.
[0159] The cDNA may then be integrated in an expression vector and
transfected into an appropriate host cell for protein expression.
The host may be a prokaryotic or eucaryotic cell since the former's
inability to glycosylate osteogenic protein will not effect the
protein's enzymatic activity. Useful host cells include
Saccharomyces, E. coli, and various mammalian cell cultures. The
vector may additionally encode various signal sequences for protein
secretion and/or may encode osteogenic protein as a fusion protein.
After being translated protein may be purified from the cells or
recovered from the culture medium.
II. Recombinant Non-Native Osteogenic Protein Constructs
[0160] A. Protein Design
[0161] This section discloses the production of novel recombinant
proteins capable of inducing cartilage and endochondral bone
comprising a protein structure duplicative of the functional domain
of the amino acid sequence encoded by consensus DNA sequences
derived from a family of natural proteins implicated in tissue
development. These gene products/proteins are known to exist in
active form as dimers and are, in general, processed from a
precursor protein to produce an active C-terminal domain of the
precursor.
[0162] The recombinant osteogenic/chondrogenic proteins are "novel"
in the sense that, as far as applicants are aware, they do not
exist in nature or, if they do exist, have never before been
associated with bone or cartilage formation. The approach to design
of these proteins was to employ amino acid sequences found in the
native isolates described above, in polypeptide structures which
are patterned after certain proteins reported in the literature, or
the amino acid sequences inferred from DNAs reported in the
literature. Thus, using the design criteria set forth above in the
probe design section, and refining the amino acid sequence as more
protein sequence information was learned, a series of synthetic
proteins were designed with the hope and intent that they might
have osteogenic or chondrogenic activity when tested in the
bioassay system disclosed below.
[0163] It was noted, for example, that DPP from drosophila, VG1
from Xenopus, the TGF beta family of proteins, and to a lesser
extent, alpha and beta inhibins, had significant homologies with
certain of the sequences derived from the naturally sourced OP
product. (FIG. 18.) Study of these proteins led to the realization
that a portion of the sequence of each had a structural similarity
observable by analysis of the positional relationship of cysteines
and other amino acids which have an important influence on three
dimensional protein conformation. It was noted that a region of
these sequences had a series of seven cysteines, placed very nearly
in the same relative positions, and certain other amino acids in
sequence as set forth below:
16 10 20 30 40 50
CXXXXLXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 60 70 80 90 100
QXXVXXXNXXXXPXXCCXPXXXXXXXXLXXXXXXXVXLXXYXXMXVXXCXCX
[0164] wherein each X independently represents an amino acid.
Expression experiments with constructs patterned after this
template amino acid sequence showed activity occurred with a
shorter sequence having only six cysteines:
17 10 20 30 40 50 LXVXFXDXGWXXWXXXPXGXXAXYCXGXCXXPXXXXXXXXNHAXX 60
70 80 90 100 QXXVXXXNXXXXPXXCCXPXXXXXXXX-
LXXXXXXXVXLXXYXXMXVXXCXCX
[0165] wherein each X independently represents an amino acid.
Within these generic structures are a multiplicity of specific
sequences which have osteogenic or chondrogenic activity. Preferred
structures are those having the amino acid sequence:
18 10 20 30 40 50
CKRHPLYVDFRDVGWNDWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIV RRRS K S S L QE
VIS E FD Y E A AY MPESMKAS VI KE F E K I DN L N S Q ITK F P TL Q A
S K 60 70 80 90 100
QTLVNSVNPGKIPKACCVPTELSAISMLYLDENENVVLKNYQDMVVEGCGCR SI HAI SEQV EP
A EQMNSLAI FFNDQDK I RK EE T DA H H RF T S K DPV V Y N S H RN RS N
S K P E
[0166] wherein, in each position where more than one amino acid is
shown, any one of the amino acids shown may be used. Novel active
proteins also are defined by amino acid sequences comprising an
active domain beginning at residue number 6 of this sequence, i.e,
omitting the N terminal CXXXX, or omitting any of the preferred
specific combinations such as CKRHP, CRRKQ, CKRHE, etc, resulting
in a construct having only 6 cysteine residues. After this work,
PCT 87/01537 was published, and it was observed that the proteins
there identified as BMPII a and b and BMPIII each comprised a
region embodying this generic structure. These proteins were not
demonstrated to be osteogenic in the published application.
However, applicants discovered that a subpart of the amino acid
sequence of these proteins, properly folded, and implanted as set
forth herein, is active. These are disclosed herein as CBMPIIa,
CBMPIIb, and CBMPIII. Also, the OP1 protein was observed to exhibit
the same generic structure.
[0167] Thus, the preferred osteogenic proteins are expressed from
recombinant DNA and comprise amino acid sequences including any of
the following sequences:
19 1 10 20 30 40 Vg1 CKKRHLYVEFK-DVGWQNWVIAPQGYMANYCYGECPYPLTE 50
60 70 ILNGSN--H-AILQTLVHSIEPED-IPLPCCVPTKMSP 80 90 100
ISMLFYDNNDNVVLRHYENMAVDECGCR 1 10 20 30 40 DPP
CRRHSLYVDFS-DVGWDDWIVAPLGYDAYYCHGKCPFPLAD 50 60 70
HFNSTN--H-AVVQTLVNNNNPGK-VPKACCVPTQLDS 80 90 100
VAMLYLNDQSTVVLKNYQEMTVVGCGCR 1 10 20 30 40 OP1
LYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA 80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH -5 HQRQA 1 10 20 30 40 OP1
CKKHELYVSFR-DLGWQDWIIAPEGYAAYYCEGECAFPLNS 50 60 70
YMNATN--H-AIVQTLVHFINPET-VPKPCCAPTQLNA 80 90 100
ISVLYFDDSSNVILKKYRNMVVRACGCH 1 10 20 30 40 CBMP-2a
CKRHPLYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 50 60 70
HLNSTN--H-AIVQTLVNSVNS-K-IPKACCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQDMVVEGCGCR 1 10 20 30 40 CBMP-2b
CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGDCPFPLAD 50 60 70
HLNSTN--H-AIVQTLVNSVNS-S-IPKACCVPTELSA 80 90 100
ISMLYLDEYDKVVLKNYQEMVVEGCGCR 1 10 20 30 40 CBMP-3
CARRYLKVDFA-DIGWSEWIISPKSFDAYYCSGACQFPMPK 50 60 70
SLKPSN--H-ATIQSIVRAVGVVPGIPEPCCVPEKMSS 80 90 100
LSILFFDENKNVVLKVYPNMTVESCACR 1 10 20 30 40 COP1
LYVDFQRDVGWDDWIIAPVDFDAYYCSGACQFPSAD 50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA 80 90 100
ISMLYLDENSTVVLKNYQEMTVVGCGCR 1 10 20 30 40 COP3
LYVDFQRDVGWDDWIVAPPGYQAFYCSGACQFPSAD 50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR 1 10 20 30 40 COP4
LYVDFS-DVGWDDWIVAPPGYQAFYCSGACQFPSAD 50 60 70
HFNSTN--H-AVVQTLVNNMNPGK-VPKPCCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR 1 10 20 30 40 COP5
LYVDFS-DVGWDDWIVAPPGYQAFYCHGECPFPLAD 50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR 1 10 20 30 40 COP7
LYVDFS-DVGWNDWIVAPPGYHAFYCHGECPFPLAD 50 60 70
HLNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR 10 PKHHSQRARKKNKN 1 10 20 30 40 COP16
CRRHSLYVDFS-DVGWNDWIVAPPGYQAFYCHGECPFPLAD 50 60 70
HFNSTN--H-AVVQTLVNSVNSKI--PKACCVPTELSA 80 90 100
ISMLYLDENEKVVLKNYQEMVVEGCGCR
[0168] As shown in FIG. 18, these sequences have considerable
homology with the alpha and beta inhibins, three forms of TGF beta,
and MIS.
[0169] B. Gene Preparation
[0170] The synthetic genes designed as described above preferably
are produced by assembly of chemically synthesized
oligonucleotides. 15-100 mer oligonucleotides may be synthesized on
a Biosearch DNA Model 8600 Synthesizer, and purified by
polyacrylamide gel electrophoresis (PAGE) in Tris-Borate-EDTA
buffer (TBE). The DNA is then electroeluted from the gel.
Overlapping oligomers may be phosphorylated by T4 polynucleotide
kinase and ligated into larger blocks which may also be purifed by
PAGE. Natural gene sequences and cDNAs also may be used for
expression.
[0171] C. Expression
[0172] The genes can be expressed in appropriate prokaryotic hosts
such as various strains of E. coli. For example, if the gene is to
be expressed in E. coli, it must first be cloned into an expression
vector. An expression vector (FIG. 21A) based on pBR322 and
containing a synthetic trp promoter operator and the modified trp
LE leader can be opened at the EcoRI and PSTI restriction sites,
and a FB-FB COP gene fragment (FIG. 21B) can be inserted between
these sites, where FB is fragment B of Staphylococcal Protein A.
The expressed fusion protein results from attachment of the COP
gene to a fragment encoding FB. The COP protein is joined to the
leader protein via a hinge region having the sequence
asp-pro-asn-gly. This hinge permits chemical cleavage of the fusion
protein with dilute acid at the asp-pro site or cleavage at asn-gly
with hydroxylamine, resulting in release of the COP protein.
[0173] D. Production of Active Proteins
[0174] The following procedure was followed for production of
active recombinant protiens. E coli cells containing the fusion
proteins were lysed. The fusion proteins were purified by
differential solubilization. In the case of the COP 1, 3, 4, 5, and
7 fusion proteins, cleavage was with dilute acid, and the resulting
cleavage products were passed through a Sephacryl-200HR column. The
Sephacryl column separated most of the uncleaved fusion products
from the COP 1, 3, 4, 5, and 7 analogs. In the case of the COP 16
fusion protein, cleavage was with a more concentrated acid, and an
SP-Trisacryl column was used to separate COP 16, the leader
protein, and the residual fusion protein. The COP fractions from
any of the COP analogs were then subjected to HPLC on a semi-prep
C-18 column. The HPLC column primarily separated the leader
proteins and other minor impurities from the COP analogs.
[0175] Initial conditions for refolding of COP analogs were at pH
8.0 using Tris, GuHCl, dithiothreitol. Final conditions for
refolding of COP analogs were at pH 8.0 using Tris, oxidized
glutathione, and lower amounts of GuHCl and dithiothreitol.
[0176] E. Production of Antisera
[0177] Antisera to COP 7 and COP5 were produced in New Zealand
white rabbits. Western blots demonstrate that the antisera react
with COP 7 and COP5 preparations. Antisera to COP 7 has been tested
for reactivity to bovine osteogenic protein samples. Western blots
show a clear reaction with the 30 kD protein and, when reduced,
with the 16 kD subunit. The immunoreactive species appears as a
closely-spared doublet in the 16K subunit region, similar to the
16K doublet seen in Con A blots.
III. Matrix Preparation
[0178] A. General Consideration of Matrix Properties
[0179] The carrier described in the bioassay section; infra, may be
replaced by either a biodegradable-synthetic or synthetic-inorganic
matrix (e.g., HAP, collagen, tricalcium phosphate, or polylactic
acid, polyglycolic acid and various copolymers thereof). Also
xenogeneic bone may be used if pretreated as described below.
[0180] Studies have shown that surface charge, particle size, the
presence of mineral, and the methodology for combining matrix and
osteogenic protein all play a role in achieving successful bone
induction. Perturbation of the charge by chemical modification
abolishes the inductive response. Particle size influences the
quantitative response of new bone; particles between 75 and 420 mm
elicit the maximum response. Contamination of the matrix with bone
mineral will inhibit bone formation. Most importantly, the
procedures used to formulate osteogenic protein onto the matrix are
extremely sensitive to the physical and chemical state of both the
osteogenic protein and the matrix.
[0181] The sequential cellular reactions at the interface of the
bone matrix/OP implants are complex. The multistep cascade
includes: binding of fibrin and fibronectin to implanted matrix,
chemotaxis of cells, proliferation of fibroblasts, differentiation
into chondroblasts, cartilage formation, vascular invasion, bone
formation, remodeling, and bone marrow differentiation.
[0182] A successful carrier for osteogenic protein must perform
several important functions. It must bind osteogenic protein and
act as a slow release delivery system, accommodate each step of the
cellular response during bone development, and protect the
osteogenic protein from nonspecific proteolysis. In addition,
selected materials must be biocompatible in vivo and biodegradable;
the carrier must act as a temporary scaffold until replaced
completely by new bone. Polylactic acid (PLA), polyglycolic acid
(PGA), and various combinations have different dissolution rates in
vivo. In bones, the dissolution rates can vary according to whether
the implant is placed in cortical or trabecular bone.
[0183] Matrix geometry, particle size, the presence of surface
charge, and porosity or the presence of interstices among the
particles of a size sufficient to permit cell infiltration, are all
important to successful matrix performance. It is preferred to
shape the matrix to the desired form of the new bone and to have
dimensions which span non-union defects. Rat studies show that the
new bone is formed essentially having the dimensions of the device
implanted.
[0184] The matrix may comprise a shape-retaining solid made of
loosely adhered particulate material, e.g., with collagen. It may
also comprise a molded, porous solid, or simply an aggregation of
close-packed particles held in place by surrounding tissue.
Masticated muscle or other tissue may also be used. Large
allogeneic bone implants can act as a carrier for the matrix if
their marrow cavities are cleaned and packed with particles and the
dispersed osteogenic protein.
[0185] B. Preparation of Biologically Active Allogenic Matrix
[0186] Demineralized bone matrix is prepared from the dehydrated
diaphyseal shafts of rat femur and tibia as described herein to
produce a bone particle size which pass through a 420 mm sieve. The
bone particles are subjected to dissociative extraction with 4 M
guanidine-HCl. Such treatment results in a complete loss of the
inherent ability of the bone matrix to induce endochondral bone
differentiation. The remaining insoluble material is used to
fabricate the matrix. The material is mostly collagenous in nature,
and upon implantation, does not induce cartilage and bone. All new
preparations are tested for mineral content and false positives
before use. The total loss of biological activity of bone matrix is
restored when an active osteoinductive protein fraction or a pure
protein is reconstituted with the biologically inactive insoluble
collagenous matrix. The osteoinductive protein can be obtained from
any vertebrate, e.g., bovine, porcine, monkey, or human, or
produced using recombinant DNA techniques.
[0187] C. Preparation of Deglycosylated Bone Matrix for Use in
Xenogenic Implant
[0188] When osteogenic protein is reconstituted with collagenous
bone matrix from other species and implanted in rat, no bone is
formed. This suggests that while the osteogenic protein is
xenogenic (not species specific), while the matrix is species
specific and cannot be implanted cross species perhaps due to
intrinsic immunogenic or inhibitory components. Thus, heretofore,
for bone-based matrices, in order for the osteogenic protein to
exhibit its full bone inducing activity, a species specific
collagenous bone matrix was required.
[0189] The major component of all bone matrices is Type I collagen.
In addition to collagen, extracted bone includes non-collagenous
proteins which may account for 5% of its mass. Many non-collagenous
components of bone matrix are glycoproteins. Although the
biological significance of the glycoproteins in bone formation is
not known, they may present themselves as potent antigens by virtue
of their carbohydrate content and may constitute immunogenic and/or
inhibitory components that are present in xenogenic matrix.
[0190] It has now been discovered that a collagenous bone matrix
may be used as a carrier to effect bone inducing activity in
xenogenic implants, if one first removes the immonogenic and
inhibitory components from the matrix. The matrix is deglycosglated
chemically using, for example, hydrogen fluoride to achieve this
purpose.
[0191] Bovine bone residue prepared as described above and
particles of the 74-420 mM are collected. The sample is dried in
vacuo over P.sub.2O.sub.5, transferred to the reaction vessel and
anhydrous hydrogen fluoride (HF) (10-20 ml/g of matrix) is then
distilled onto the sample at -70.degree. C. The vessel is allowed
to warm to 0.degree. and the reaction mixture is stirred at this
temperature for 60 min. After evaporation of the HF in vacuo, the
residue is dried thoroughly in vacuo over KOH pellets to remove any
remaining traces of acid.
[0192] Extent of deglycosylation can be determined from
carbohydrate analysis of matrix samples taken before and after
treatment with HF, after washing the samples appropriately to
remove non-covalently bound carbohydrates.
[0193] The deglycosylated bone matrix is next treated as set forth
below:
[0194] 1) suspend in TBS (Tris-buffered Saline) 1 g/200 ml and stir
at 4.degree. C. for 2 hrs;
[0195] 2) centrifuge then treated again with TBS, 1 g/200 ml and
stir at 4.degree. C. overnight; and
[0196] 3) centrifuged; discard supernatant; water wash residue; and
then lyophilized.
IV. Fabrication of Device
[0197] Fabrication of osteogenic devices using any of the matrices
set forth above with any of the osteogenic proteins described above
may be performed as follows.
[0198] A. Ethanol Precipitation
[0199] In this procedure, matrix was added to osteogenic protein in
guanidine-HCl. Samples were vortexed and incubated at a low
temperature. Samples were then further vortexed. Cold absolute
ethanol was added to the mixture which was then stirred and
incubated. After centrifugation (microfuge high speed) the
supernatant was discarded. The reconstituted matrix was washed with
cold concentrated ethanol in water and then lyophilized.
[0200] B. Acetonitrile Trifluoroacetic Acid Lyophilization
[0201] In this procedure, osteogenic protein in an acetonitrile
trifluroacetic acid (ACN/TFA) solution was added to the carrier.
Samples were vigorously vortexed many times and then lyophilized.
Osteogenic protein was added in varying concentrations obtained at
several levels of purity that have been tested to determine the
most effective dose/purity level in rat in vivo assay.
[0202] C. Urea Lyophilization
[0203] For those proteins that are prepared in urea buffer, the
protein is mixed with the matrix, vortexed many times, and then
lyophilized. The lyophilized material may be used "as is" for
implants.
V. In Vivo Rat Bioassay
[0204] Substantially pure BOP, BOP-rich extracts comprising protein
having the properties set forth above, and several of the synthetic
proteins have been incorporated in matrices to produce osteogenic
devices, and assayed in rat for endochondral bone. Studies in rats
show the osteogenic effect to be dependent on the dose of
osteogenic protein dispersed in the osteogenic device. No activity
is observed if the matrix is implanted alone. The following sets
forth guidelines for how the osteogenic devices disclosed herein
might be assayed for determining active fractions of osteogenic
protein when employing the isolation procedure of the invention,
and evaluating protein constructs and matrices for biological
activity.
[0205] A. Subcutaneous Implantation
[0206] The bioassay for bone induction as described by Sampath and
Reddi (Proc. Natl. Acad. Sci. USA (1983) 80: 6591-6595), herein
incorporated by reference, is used to monitor the purification
protocols for endochondral bone differentiation activity. This
assay consists of implanting the test samples in subcutaneous sites
in allogeneic recipient rats under ether anesthesia. Male
Long-Evans rats, aged 28-32 days, were used. A vertical incision (1
cm) is made under sterile conditions in the skin over the thoraic
region, and a pocket is prepared by blunt dissection. Approximately
25 mg of the test sample is implanted deep into the pocket and the
incision is closed with a metallic skin clip. The day of
implantation is designated as day of the experiment. Implants were
removed on day 12. The heterotropic site allows for the study of
bone induction without the possible ambiguities resulting from the
use of orthotopic sites.
[0207] B. Cellular Events
[0208] The implant model in rats exhibits a controlled progression
through the stages of matrix induced endochondral bone development
including: (1) transient infiltration by polymorphonuclear
leukocytes on day one; (2) mesenchymal cell migration and
proliferation on days two and three; (3) chondrocyte appearance on
days five and six; (4) cartilage matrix formation on day seven; (5)
cartiliage calcification on day eight; (6) vascular invasion,
appearance of osteoblasts, and formation of new bone on days nine
and ten; (7) appearance of osteoblastic and bone remodeling and
dissolution of the implanted matrix on days twelve to eighteen; and
(8) hematopoietic bone marrow differentiation in the ossicle on day
twenty-one. The results show that the shape of the new bone
conforms to the shape of the implanted matrix.
[0209] C. Histological Evaluation
[0210] Histological sectioning and staining is preferred to
determine the extent of osteogenesis in the implants. Implants are
fixed in Bonus Solution, embedded in parafilm, cut into 6-8 mm
sections. Staining with toluidine blue or hemotoxylin/eosin
demonstrates clearly the ultimate development of endochondrial
bone. Twelve day implants are usually sufficient to determine
whether the implants show bone inducing activity.
[0211] D. Biological Markers
[0212] Alkaline phosphatase activity may be used as a marker for
osteogenesis. The enzyme activity may be determined
spectrophotometrically after homogenization of the implant. The
activity peaks at 9-10 days in vivo and thereafter slowly declines.
Implants showing no bone development by histology should have
little or no alkaline phosphatase activity under these assay
conditions. The assay is useful for quantitation and obtaining an
estimate of bone formation very quickly after the implants are
removed from the rat. In order to estimate the amount of bone
formation, the calcium content of the implant is determined.
[0213] Implants containing osteogenic protein at several levels of
purity have been tested to determine the most effective dose/purity
level, in order to seek a formulation which could be produced on an
industrial scale. The results as measured by specific acivity of
alkaline phosphatase and calcium content, and histological
examination. For specific activity of alkaline phosphatase is
elevated during onset of bone formation and then declines. On the
other hand, calcium content is directly proportional to the total
amount of bone that is formed. The osteogenic activity due to
osteogenic protein is represented by "bone forming units". For
example, one bone forming unit represents the amount of protein
that is needed for half maximal bone forming activity as compared
to rat demineralized bone matrix as control and determined by
calcium content of the implant on day 12.
[0214] E. Results
[0215] E-1. Natural Sourced Osteogenic Protein
[0216] Dose curves are constructed for bone inducing activity in
vivo at each step of the purification scheme by assaying various
concentrations of protein. FIG. 11 shows representative dose curves
in rats as determined by alkaline phosphatase. Similar results are
obtained when represented as bone forming units. Approximately
10-12 mg of the TSK-fraction, 3-4 mg of heparin-Sepharose-II
fraction, 0.4-0.5 mg of the C-18 column purified fraction, and
20-25 ng of gel eluted highly purified 30 kD protein is needed for
unequivocal bone formation (half maximum activity). 20-25 ng per 25
mg of implant is normally sufficient to produce endochondral bone.
Thus, 1-2 ng osteogenic protein per mg of implant is a reasonable
dosage, although higher dosages may be used. (See section IBS on
specific activity of osteogenic protein.)
E-2. Xenogenic Matrix Results
[0217] Deglycosylated xenogenic collagenous bone matrix (example:
bovine) has been used instead of allogenic collagenous matrix to
prepare osteogenic devices (see previous section) and bioassayed in
rat for bone inducing activity in vivo. The results demonstrate
that xenogenic collagenous bone matrix after chemical
deglycosylation induces successful endochondral bone formation
(FIG. 19). As shown by specific activity of alkaline phosphotase,
it is evident that the deglycosylated xenogenic matrix induced bone
whereas untreated bovine matrix did not.
[0218] Histological evaluation of implants suggests that the
deglycosylated bovine matrix not only has induced bone in a way
comparable to the rat residue matrix but also has advanced the
developmental stages that are involved in endochondral bone
differentiation. Compared to rat residue as control, the HF treated
bovine matrix contains extensively remodeled bone. Ossicles are
formed that are already filled with bone marrow elements by 12
days. This profound action as elicited by deglycosylated bovine
matrix in supporting bone induction is reproducible and is dose
dependent with varying concentration of osteogenic protein.
[0219] E-3. Synthetic/Recombinant Proteins (COP5, COP7)
[0220] The device that contained only rat carrier showed complete
absence of new bone formation. The implant consists of carrier rat
matrix and surrounding mesenchymal cells. Again, the devices that
contained rat carrier and not correctly folded (or biologically
inactive) recombinant protein also showed complete absence of bone
formation. These implants are scored as cartilage formation (-) and
bone formation (-). The endochondral bone formation activity is
scored as zero percent (0%). (FIG. 22A)
[0221] Implants included biologically active recombinant protein,
however, showed evidence of endochondral bone formation.
Histologically they showed new cartilage and bone formation.
[0222] The cartilage formation is scored as (+) by the presence of
metachromatically stained chondrocytes in center of the implant, as
(++) by the presence of numerous chondrocytes in many areas of the
implant and as (+++) by the presence of abundant chondrocytes
forming cartilage matrix and the appearance of hypertrophied
chondrocytes accompanying cartilage calcification (FIG. 22B).
[0223] The bone formation is scored as (+) by the presence of
osteoblast surrounding vascular endothelium forming new matrix, and
as (++) by the formation of bone due to osteoblasts (as indicated
by arrows) and further bone remodeling by the appearance of
osteoblasts in apposition to the rat carrier. Vascular invasion is
evident in these implants (FIG. 22B).
[0224] The overall bone inducing activity due to recombinant
protein is represented as percent response of endochondral bone
formation (see Table 7 below) The percent response means the area
of the implant that is covered by newly induced cartilage and bone
as shown by histology in low magnification.
20TABLE 7 HISTOLOGICAL EVALUATION OF RECOMBINANT BONE INDUCTIVE
PROTEINS Percent Implanted Cartilage Bone Response in Protein
Formation Formation the Implant COP-5 +++ ++ 15% COP-5 ++ + 5%
COP-7 +++ ++ 30% COP-7 +++ ++ 20% COP-7 ++ + 20% COP-7 ++ + 10%
COP-7 +++ ++ 30% COP-7 ++ ++ 20% COP-5 +++ ++ 20%
VI. Animal Efficacy Studies
[0225] Substantially pure osteogenic protein from bovine bone
(BOP), BOP-rich osteogenic fractions having the properties set
forth above, and several of the synthetic/recombinant proteins have
been incorporated in matrices to produce osteogenic devices. The
efficacy of bone-inducing potential of these devices was tested in
cat and rabbit models, and found to be potent inducers of
osteogenesis, ultimately resulting in formation of mineralized
bone. The following sets forth guidelines as to how the osteogenic
devices disclosed herein might be used in a clinical setting.
[0226] A. Feline Model
[0227] The purpose of this study is to establish a large animal
efficacy model for the testing of the osteogenic devices of the
invention, and to characterize repair of massive bone defects and
simulated fracture non-union encountered frequently in the practice
of orthopedic surgery. The study is designed to evaluate whether
implants of osteogenic protein with a carrier can enhance the
regeneration of bone following injury and major reconstructive
surgery by use of this large mammal model. The first step in this
study design consists of the surgical preparation of a femoral
osteotomy defect which, without further intervention, would
consistently progress to non-union of the simulated fracture
defect. The effects of implants of osteogenic devices into the
created bone defects were evaluated by the following study
protocol.
[0228] A-1. Procedure
[0229] Sixteen adult cats weighing less than 10 lbs. undergo
unilateral preparation of a 1 cm bone defect in the right femur
through a lateral surgical approach. In other experiments, a 2 cm
bone defect was created. The femur is immediately internally fixed
by lateral placement of an 8-hole plate to preserve the exact
dimensions of the defect. There are three different types of
materials implanted in the surgically created cat femoral defects:
group I (n=3) is a control group which undergo the same plate
fixation with implants of 4 M guanidine-HCl-treated (inactivated)
cat demineralized bone matrix powder (GuHCl-DBM) (360 mg); group II
(n=31 is a positive control group implanted with biologically
active demineralized bone matrix powder (DBM) (360 mg); and group
III (n=10) undergo a procedure identical to groups I-II, with the
addition of osteogenic protein onto each of the GuHCl-DBM carrier
samples. To summarize, the group III osteogenic protein-treated
animals are implanted with exactly the same material as the group
II animals, but with the singular addition of osteogenic
protein.
[0230] All animals are allowed to ambulate ad libitum within their
cages post-operatively. All cats are injected with tetracycline (25
mg/kg SQ each week for four weeks) for bone labelling. All but four
group III animals are sacrificed four months after femoral,
osteotomy.
[0231] A-2. Radiomorphometrics
[0232] In vivo radiomorphometric studies are carried out
immediately post-op at 4, 8, 12 and 16 weeks by taking a
standardized x-ray of the lightly anesthesized animal positioned in
a cushioned x-ray jig designed to consistently produce a true
anterio-posterior view of the femur and the osteotomy site. All
x-rays are taken in exactly the same fashion and in exactly the
same position on each animal. Bone repair is calculated as a
function of mineralization by means of random point analysis. A
final specimen radiographic study of the excised bone is taken in
two planes after sacrifice. X-ray results are shown in FIG. 12, and
displaced as percent of bone defect repair. To summarize, at 16
weeks, 60% of the group III femors are united with average 86% bone
defect regeneration. By contrast, the group I GuHCl-DMB
negative-control implants exhibit no bone growth at four weeks,
less than 10% at eight and 12 weeks, and 16% (t 10%) at 16 weeks
with one of the five exhibiting a small amount of bridging bone.
The group II DMB positive-control implants exhibited 18% (.+-.3%)
repair at four weeks, 35% at eight weeks, 50% (.+-.10%) at twelve
weeks and 70% (.+-.12%) by 16 weeks, a statistical difference of
p<0.01 compared to osteogenic protein at every month. One of the
three (33%) is united at 16 weeks.
[0233] A-3. Biomechanics
[0234] Excised test and normal femurs are immediately studied by
bone densitometry, wrapped in two layers of saline-soaked towels,
placed in two sealed plastic bags, and stored at -20.degree. C.
until further study. Bone repair strength, load to failure, and
work to failure are tested by loading to failure on a specially
designed steel 4-point bending jig attached to an Instron testing
machine to quantitate bone strength, stiffness, energy absorbed and
deformation to failure. The study of test femurs and normal femurs
yield the bone strength (load) in pounds and work to failure in
joules. Normal femurs exhibit a strength of 96 (.+-.12) pounds
osteogenic protein-implanted femurs exhibited 35 (+4) pounds, but
when corrected for surface area at the site of fracture (Due to the
"hourglass" shape of the bone defect repair) this correlated
closely with normal bone strength. Only one demineralized bone
specimen was available for testing with a strength of 25 pounds,
but, again, the strength correlated closely with normal bone when
corrected for fracture surface area.
[0235] A-4. Histomorphometry/Histology
[0236] Following biomechanical testing the bones are immediately
sliced into two longitudinal sections at the defect site, weighed,
and the volume measured. One-half is fixed for standard calcified
bone histomorphometrics with fluorescent stain incorporation
evaluation, and one-half is fixed for decalcified hemotoxylin/eosin
stain histology preparation.
[0237] A-5. Biochemistry
[0238] Selected specimens from the bone repair site (n=6) are
homogenized in cold 0.15 M NaCl, 3 mM NaHCO.sub.3, pH 9.0 by a Spex
freezer mill. The alkaline phosphatase activity of the supernatant
and total calcium content of the acid soluble fraction of sediment
are then determined.
[0239] A-6. Histopathology
[0240] The final autopsy reports reveal no unusual or pathologic
findings noted at necropsy of any of the animals studied. Portion
of all major organs are preserved for further study. A
histopathological evaluation is performed on samples of the
following organs: heart, lung, liver, both kidneys, spleen, both
adrenals, lymph nodes, left and right quadriceps muscles at
mid-femur (adjacent to defect site in experimental femur). No
unusual or pathological lesions are seen in any of the tissues.
Mild lesions seen in the quadriceps muscles are compatible with
healing responses to the surgical manipulation at the defect site.
Pulmonary edema is attributable to the euthanasia procedure. There
is no evidence of any general systemic effects or any effects on
the specific organs examined.
[0241] A-7. Feline Study Summary
[0242] The 1 cm and 2 cm femoral defect cat studies demonstrate
that devices comprising a matrix containing disposed osteogenic
protein can: (1) repair a weight-bearing bone defect in a large
animal; (2) consistently induces bone formation shortly following
(less than two weeks) implantation; and (3) induce bone by
endochondral ossification, with a strength equal to normal bone, on
a volume for volume basis. Furthermore, all animals remained
healthy during the study and showed no evidence of clinical or
histological laboratory reaction to the implanted device. In this
bone defect model, there was little or no healing at control bone
implant sites. The results provide evidence for the successful use
of osteogenic devices to repair large, non-union bone defects.
[0243] B. Rabbit Model:
[0244] B1. Procedure and Results
[0245] Eight mature (less than 10 lbs) New Zealand White rabbits
with epiphyseal closure documented by X-ray were studied. The
purpose of this study is to establish a model in which there is
minimal or no bone growth in the control animals, so that when bone
induction is tested, only a strongly inductive substance will yield
a positive result. Defects of 1.5 cm are created in the rabbits,
with implantation of: osteogenic protein (n=5), DBM (n=8),
GuHCl-DBM (n=6), and no implant (n=10). Six osteogenic protein
implants are supplied and all control defects have no implant
placed.
[0246] Of the eight animals (one animal each was sacrificed at one
and two weeks), 11 ulnae defects are followed for the full course
of the eight week study. In all cases (n=7) following
osteo-periosteal bone resection, the no implant animals establish
no radiographic union by eight weeks. All no implant animals
develop a thin "shell" of bone growing from surrounding bone
present at four weeks and, to a slightly greater degree, by eight
weeks. In all cases (n=4), radiographic union with marked bone
induction is established in the osteogenic protein-implanted
animals by eight weeks. As opposed to the no implant repairs, this
bone repair is in the site of the removed bone.
[0247] Radiomorphometric analysis reveal 90% osteogenic
protein-implant bone repair and 18% no-implant bone repair at
sacrifice at eight weeks. At autopsy, the osteogenic protein bone
appears normal, while "no implant" bone sites have only a soft
fibrous tissue with no evidence or cartilage or bone repair in the
defect site.
[0248] B-2. Allograft Device
[0249] In another experiment, the marrow cavity of the 1.5 cm ulnar
defect is packed with activated osteogenic protein rabbit bone
powder and the bones are allografted in an intercalary fashion. The
two control ulnae are not healed by eight weeks and reveal the
classic "ivory" appearance. In distinct contrast, the osteogenic
protein-treated implants "disappear" radiographically by four weeks
with the start of remineralization by six to eight weeks. These
allografts heal at each end with mild proliferative bone formation
by eight weeks.
[0250] This type of device serves to accelerate allograph
repair.
[0251] B-3. Summary
[0252] These studies of 1.5 cm osteo-periosteal defects in the
ulnae of mature rabbits show that: (1) it is a suitable model for
the study of bone growth; (2) no implants or GuHCl negative control
implants yield a small amount of periosteal-type bone, but not
medullary or cortical bone growth; (3) osteogenic protein-implanted
rabbits exhibited proliferative bone growth in a fashion highly
different from the control groups; (4) initial studies show that
the bones exhibit 50% of normal bone strength (100% of normal
correlated vol:vol) at only eight weeks after creation of the
surgical defect; and (5) osteogenic protein-allograft studies
reveal a marked effect upon both the allograft and bone
healing.
[0253] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiments are therefore to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
Sequence CWU 1
1
72 1 96 PRT Artificial Sequence Biosynthetic Protein COP5 1 Leu Tyr
Val Asp Phe Ser Asp Val Gly Trp Asp Asp Trp Ile Val Ala 1 5 10 15
Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly Glu Cys Pro Phe Pro 20
25 30 Leu Ala Asp His Phe Asn Ser Thr Asn His Ala Val Val Gln Thr
Leu 35 40 45 Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala Cys Cys
Val Pro Thr 50 55 60 Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp
Glu Asn Glu Lys Val 65 70 75 80 Val Leu Lys Asn Tyr Gln Glu Met Val
Val Glu Gly Cys Gly Cys Arg 85 90 95 2 96 PRT Artificial Sequence
Biosynthetic protein COP7 2 Leu Tyr Val Asp Phe Ser Asp Val Gly Trp
Asn Asp Trp Ile Val Ala 1 5 10 15 Pro Pro Gly Tyr His Ala Phe Tyr
Cys His Gly Glu Cys Pro Phe Pro 20 25 30 Leu Ala Asp His Leu Asn
Ser Thr Asn His Ala Val Val Gln Thr Leu 35 40 45 Val Asn Ser Val
Asn Ser Lys Ile Pro Lys Ala Cys Cys Val Pro Thr 50 55 60 Glu Leu
Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val 65 70 75 80
Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu Gly Cys Gly Cys Arg 85
90 95 3 97 PRT Artificial Sequence endochondral bone formation
inducing protein 3 Leu Xaa Val Xaa Phe Xaa Asp Xaa Gly Trp Xaa Xaa
Trp Xaa Xaa Xaa 1 5 10 15 Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa
Gly Xaa Cys Xaa Xaa Pro 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Asn His Ala Xaa Xaa Gln Xaa Xaa 35 40 45 Val Xaa Xaa Xaa Asn Xaa
Xaa Xaa Xaa Pro Xaa Xaa Cys Cys Xaa Pro 50 55 60 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Val Xaa
Leu Xaa Xaa Tyr Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 85 90 95
Xaa 4 102 PRT Artificial Sequence endochondral bone formation
inducing protein 4 Cys Xaa Xaa Xaa Xaa Leu Xaa Val Xaa Phe Xaa Asp
Xaa Gly Trp Xaa 1 5 10 15 Xaa Trp Xaa Xaa Xaa Pro Xaa Gly Xaa Xaa
Ala Xaa Tyr Cys Xaa Gly 20 25 30 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Asn His Ala 35 40 45 Xaa Xaa Gln Xaa Xaa Val
Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Pro Xaa 50 55 60 Xaa Cys Cys Xaa
Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 80 Xaa Xaa
Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Tyr Xaa Xaa Met Xaa Val 85 90 95
Xaa Xaa Cys Xaa Cys Xaa 100 5 97 PRT Artificial Sequence
endochondral bone formation inducing protein 5 Leu Xaa Val Xaa Phe
Xaa Asp Xaa Gly Trp Xaa Xaa Trp Xaa Xaa Xaa 1 5 10 15 Pro Xaa Gly
Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 20 25 30 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Gln Xaa Xaa 35 40
45 Val Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Pro Xaa Xaa Cys Cys Xaa Pro
50 55 60 Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa Xaa
Xaa Xaa 65 70 75 80 Xaa Val Xaa Lys Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa
Xaa Gly Xaa Arg 85 90 95 His 6 101 PRT Artificial Sequence
endochondral bone formation inducing protein 6 Cys Xaa Xaa Xaa Xaa
Leu Xaa Asp Phe Xaa Asp Xaa Gly Trp Xaa Xaa 1 5 10 15 Trp Xaa Xaa
Xaa Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa 20 25 30 Cys
Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa 35 40
45 Xaa Gln Xaa Xaa Val Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Pro Xaa Xaa
50 55 60 Cys Cys Xaa Pro Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr
Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Val Xaa Lys Xaa Xaa Xaa Xaa
Xaa Val Xaa Xaa 85 90 95 Xaa Xaa Gly Xaa Arg 100 7 102 PRT
Artificial Sequence Vg1 protein sequence with osteogenic activity 7
Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys Asp Val Gly Trp Gln 1 5
10 15 Asn Trp Val Ile Ala Pro Gln Gly Tyr Met Ala Asn Tyr Cys Tyr
Gly 20 25 30 Glu Cys Pro Tyr Pro Leu Thr Glu Ile Leu Asn Gly Ser
Asn His Ala 35 40 45 Ile Leu Gln Thr Leu Val His Ser Ile Glu Pro
Glu Asp Ile Pro Leu 50 55 60 Pro Cys Cys Val Pro Thr Lys Met Ser
Pro Ile Ser Met Leu Phe Tyr 65 70 75 80 Asp Asn Asn Asp Asn Val Val
Leu Arg His Tyr Glu Asn Met Ala Val 85 90 95 Asp Glu Cys Gly Cys
Arg 100 8 102 PRT Artificial Sequence DPP protein sequence with
osteogenic activity 8 Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser
Asp Val Gly Trp Asp 1 5 10 15 Asp Trp Ile Val Ala Pro Leu Gly Tyr
Asp Ala Tyr Tyr Cys His Gly 20 25 30 Lys Cys Pro Phe Pro Leu Ala
Asp His Phe Asn Ser Thr Asn His Ala 35 40 45 Val Val Gln Thr Leu
Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys 50 55 60 Ala Cys Cys
Val Pro Thr Gln Leu Asp Ser Val Ala Met Leu Tyr Leu 65 70 75 80 Asn
Asp Gln Ser Thr Val Val Leu Lys Asn Tyr Gln Glu Met Thr Val 85 90
95 Val Gly Cys Gly Cys Arg 100 9 107 PRT Artificial Sequence OP1
protein sequence with osteogenic activity 9 His Gln Arg Gln Ala Cys
Lys Lys His Glu Leu Tyr Val Ser Phe Arg 1 5 10 15 Asp Leu Gly Trp
Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 20 25 30 Tyr Tyr
Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 35 40 45
Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 50
55 60 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala
Ile 65 70 75 80 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu
Lys Lys Tyr 85 90 95 Arg Asn Met Val Val Arg Ala Cys Gly Cys His
100 105 10 103 PRT Artificial Sequence CBP-2a protein sequence with
osteogenic activity 10 Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser
Asp Val Gly Trp Asn 1 5 10 15 Asp Trp Ile Val Ala Pro Pro Gly Tyr
His Ala Phe Tyr Cys His Gly 20 25 30 Glu Cys Pro Phe Pro Leu Ala
Asp His Leu Asn Ser Thr Asn His Ala 35 40 45 Ile Val Gln Thr Leu
Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala 50 55 60 Cys Cys Val
Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Tyr 65 70 75 80 Leu
Asp Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gln Asp Met Val 85 90
95 Val Glu Gly Cys Gly Cys Arg 100 11 100 PRT Artificial Sequence
CBMP-2b protein sequence with osteogenic activity 11 Cys Arg Arg
His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 1 5 10 15 Asp
Trp Ile Val Ala Pro Pro Gly Tyr Gln Ala Phe Tyr Cys His Gly 20 25
30 Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala
35 40 45 Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Ile Pro Lys
Ala Cys 50 55 60 Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu
Tyr Leu Asp Glu 65 70 75 80 Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln
Glu Met Val Val Glu Gly 85 90 95 Cys Gly Cys Arg 100 12 103 PRT
Artificial Sequence CBMP-3 12 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 13 98 PRT Artificial
Sequence COP1 13 Leu Tyr Val Asp Phe Gln Arg Asp Val Gly Trp Asp
Asp Trp Ile Ile 1 5 10 15 Ala Pro Val Asp Phe Asp Ala Tyr Tyr Cys
Ser Gly Ala Cys Gln Phe 20 25 30 Pro Ser Ala Asp His Phe Asn Ser
Thr Asn His Ala Val Val Gln Thr 35 40 45 Leu Val Asn Asn Met Asn
Pro Gly Lys Val Pro Lys Pro Cys Cys Val 50 55 60 Pro Thr Glu Leu
Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Ser 65 70 75 80 Thr Val
Val Leu Lys Asn Tyr Gln Glu Met Thr Val Val Gly Cys Gly 85 90 95
Cys Arg 14 98 PRT Artificial Sequence COP3 14 Leu Tyr Val Asp Phe
Gln Arg Asp Val Gly Trp Asp Asp Trp Ile Val 1 5 10 15 Ala Pro Pro
Gly Tyr Gln Ala Phe Tyr Cys Ser Gly Ala Cys Gln Phe 20 25 30 Pro
Ser Ala Asp His Phe Asn Ser Thr Asn His Ala Val Val Gln Thr 35 40
45 Leu Val Asn Asn Met Asn Pro Gly Lys Val Pro Lys Pro Cys Cys Val
50 55 60 Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu
Asn Glu 65 70 75 80 Lys Val Val Leu Lys Asn Tyr Gln Glu Met Val Val
Glu Gly Cys Gly 85 90 95 Cys Arg 15 97 PRT Artificial Sequence COP4
15 Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp Asp Trp Ile Val Ala
1 5 10 15 Pro Pro Gly Tyr Gln Ala Phe Tyr Cys Ser Gly Ala Cys Gln
Phe Pro 20 25 30 Ser Ala Asp His Phe Asn Ser Thr Asn His Ala Val
Val Gln Thr Leu 35 40 45 Val Asn Asn Met Asn Pro Gly Lys Val Pro
Lys Pro Cys Cys Val Pro 50 55 60 Thr Glu Leu Ser Ala Ile Ser Met
Leu Tyr Leu Asp Glu Asn Glu Lys 65 70 75 80 Val Val Leu Lys Asn Tyr
Gln Glu Met Val Val Glu Gly Cys Gly Cys 85 90 95 Arg 16 97 PRT
Artificial Sequence COP16 16 Leu Tyr Val Asp Phe Ser Asp Val Gly
Trp Asp Asp Trp Ile Val Ala 1 5 10 15 Pro Pro Gly Tyr Gln Ala Phe
Tyr Cys Ser Gly Ala Cys Gln Phe Pro 20 25 30 Ser Ala Asp His Phe
Asn Ser Thr Asn His Ala Val Val Gln Thr Leu 35 40 45 Val Asn Asn
Met Asn Pro Gly Lys Val Pro Lys Pro Cys Cys Val Pro 50 55 60 Thr
Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys 65 70
75 80 Val Val Leu Lys Asn Tyr Gln Glu Met Val Val Glu Gly Cys Gly
Cys 85 90 95 Arg 17 17 PRT Artificial Sequence peptide fragment 17
Ser Phe Asp Ala Tyr Tyr Cys Ser Gly Ala Cys Gln Phe Pro Met Pro 1 5
10 15 Lys 18 14 PRT Artificial Sequence peptide sequence 18 Ser Leu
Lys Pro Ser Asn Tyr Ala Thr Ile Gln Ser Ile Val 1 5 10 19 21 PRT
Artificial Sequence peptide fragment 19 Ala Cys Cys Val Pro Thr Glu
Leu Ser Ala Ile Ser Met Leu Tyr Leu 1 5 10 15 Asp Glu Asn Glu Lys
20 20 13 PRT Artificial Sequence peptide fragment 20 Met Ser Ser
Leu Ser Ile Leu Phe Phe Asp Glu Asn Lys 1 5 10 21 10 PRT Artificial
Sequence peptide fragment 21 Ser Gln Glu Leu Tyr Val Asp Phe Gln
Arg 1 5 10 22 11 PRT Artificial Sequence peptide fragment 22 Phe
Leu His Cys Gln Phe Ser Glu Arg Asn Ser 1 5 10 23 15 PRT Artificial
Sequence peptide fragment 23 Thr Val Gly Gln Leu Asn Glu Gln Ser
Ser Glu Pro Asn Ile Tyr 1 5 10 15 24 7 PRT Artificial Sequence
peptide fragment 24 Leu Tyr Asp Pro Met Val Val 1 5 25 15 PRT
Artificial Sequence peptide fragment 25 Val Gly Val Val Pro Gly Ile
Pro Glu Pro Cys Cys Val Pro Glu 1 5 10 15 26 7 PRT Artificial
Sequence peptide fragment 26 Val Asp Phe Ala Asp Ile Gly 1 5 27 9
PRT Artificial Sequence peptide fragment 27 Val Pro Lys Pro Cys Cys
Ala Pro Thr 1 5 28 7 PRT Artificial Sequence peptide fragment 28
Ile Asn Ile Ala Asn Tyr Leu 1 5 29 13 PRT Artificial Sequence
peptide fragment 29 Asp Asn His Val Leu Thr Met Phe Pro Ile Ala Ile
Asn 1 5 10 30 16 PRT Artificial Sequence peptide fragment 30 Asp
Glu Gln Thr Leu Lys Lys Ala Arg Arg Lys Gln Trp Ile Xaa Pro 1 5 10
15 31 11 PRT Artificial Sequence peptide fragment 31 Asp Ile Gly
Xaa Ser Glu Trp Ile Ile Xaa Pro 1 5 10 32 17 PRT Artificial
Sequence peptide fragment 32 Ser Ile Val Arg Ala Val Gly Val Pro
Gly Ile Pro Glu Pro Xaa Xaa 1 5 10 15 Val 33 13 PRT Artificial
Sequence peptide fragment 33 Asp Xaa Ile Val Ala Pro Pro Gln Tyr
His Ala Phe Tyr 1 5 10 34 17 PRT Artificial Sequence peptide
fragment 34 Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met
Thr Val 1 5 10 15 Glu 35 18 PRT Artificial Sequence peptide
fragment 35 Ser Gln Thr Leu Gln Phe Asp Glu Gln Thr Leu Lys Xaa Ala
Arg Xaa 1 5 10 15 Lys Gln 36 24 PRT Artificial Sequence peptide
fragment 36 Asp Glu Gln Thr Leu Lys Lys Ala Arg Arg Lys Gln Trp Ile
Glu Pro 1 5 10 15 Arg Asn Xaa Ala Arg Arg Tyr Leu 20 37 20 PRT
Artificial Sequence peptide fragment 37 Ala Arg Arg Lys Gln Trp Ile
Glu Pro Arg Asn Xaa Ala Xaa Arg Tyr 1 5 10 15 Xaa Xaa Val Asp 20 38
23 PRT Artificial Sequence peptide fragment 38 Arg Xaa Gln Trp Ile
Glu Pro Xaa Asn Xaa Ala Xaa Xaa Tyr Leu Lys 1 5 10 15 Val Asp Xaa
Ala Xaa Xaa Gly 20 39 97 PRT Artificial Sequence OP1 shorter
sequence 39 Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile
Ile Ala 1 5 10 15 Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu
Cys Ala Phe Pro 20 25 30 Leu Asn Ser Tyr Met Asn Ala Thr Asn His
Ala Ile Val Gln Thr Leu 35 40 45 Val His Phe Ile Asn Pro Glu Thr
Val Pro Lys Pro Cys Cys Ala Pro 50 55 60 Thr Gln Leu Asn Ala Ile
Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn 65 70 75 80 Val Ile Leu Lys
Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys 85 90 95 His 40
4805 DNA Artificial Sequence genomic sequence of OP1 40 ggaggtatag
gagctctctt cgattttagc aaaccaggag tccgaagatc taaggagagc 60
tgggggtttg actccgagag ctcgagcagt ccccaagacc tggtcttgac tcacgagtta
120 gactccactc agaggctgac tgtctccagg gtctacacct ctaagggcga
cactgggctc 180 aagcagactg ccgttttcta tatgggatga gccttcacag
ggcagccagt tgggatgggt 240 tgaggtttgg ctgtagacat cagaaaccca
agtcaaatgc gcttcaacca gtagaaaatt 300 caccagcccg cagagctaag
gttgggtgga cattagggtt ggttgatcca ggagctcaac 360 agtgtcctct
gagccccagc tccttctgcc ccaccccacc atcttcagtg ctgcttcctc 420
tcaaggccac agctgtagtt ggccaggggg gcttcattat tttttgctcc tgggcagtag
480 gaggaagaga atgaatgtct ctccatgggt ctttcttagg aatgtgggaa
ctttttccag 540 aagtctctat gtcttttagt ttgtgttggg tcacttgccc
ttcctgaacc acttcctgac 600 tcctggacag gatgtgcact gatgagctta
gctttgggga tctaatagtg actttacaaa 660 gcctctttga gaaggtgaca
ttggaaccaa ggcttgagca gacacaacaa agattgcagg 720 gaggggcatt
gcaggtggag gaaacggcac atgcaagagc cctgcgtggg agtgagcttg 780
gtgtttggtc aatcagttgt cagagcacac cgggccctgt cagcaggcac agcctgggcc
840 tgctctgagt atgacagaga gcccctggga
agttgtaggt ggaggaaaga caggtcatga 900 ctaggaaaaa agcaatccct
ctgttgtggg gtggaaggaa ggttgcagtg tgtgtgagag 960 agagacaaga
cagacagaca gacacttctc aatgtttaca agtgctcagg ccctgacccg 1020
aatgcttcca aatttacgta gttctggaaa accccctgta tcattttcac tactcaaaga
1080 aacctcggga gtgttttctt ctgaaaggtc atcaggtttt gactctctgc
tgtctcattt 1140 cttcttgctg gtggtggtga tggttgcttg tcccaggccc
tgtcccgcat cctcttgccc 1200 ctgcagaggg atgagtgtgt tggggcctca
cgagttgagg ttgttcataa gcagatctct 1260 ttgagcaggg cgcctgcagt
ggccttgtgt gaggctggag gggtttcgat tcccttatgg 1320 aatccaggca
gatgtagcat ttaaacaaca cacgtgtata aaagaaacca gtgtccgcag 1380
aaggttccag aaagtattat gggataagac tacatgagag aggaatgggg cattggcacc
1440 tcccttagta gggcctttgc tgggggtaga aatgagtttt aaggcaggtt
agaccctcga 1500 actggctttt gaatcgggaa atttaccccc cagccgttct
gtgcttcatt gctgttcaca 1560 tcactgccta agatggagga actttgatgt
gtgtgtgttt ctttctcctc actgggctct 1620 gcttcttcac ttccttgtca
atgcagagaa cagcagcagg caccagaggc aggccttgta 1680 agaagcacga
gctgtatgtc agcttccgag acctgggctg gcaggtaagg ggctggctgg 1740
gtctgtcttg ggtgtgggcc ctctggcgtg ggctcccaca ggcagcgggt gctgtgctca
1800 gtcttgtttc tcatctctgc cagttaagac tccagtatca agtggcctcg
ctagggaagg 1860 gtacttggct aaggatacag gggggagcca gcatgggtga
tgccattatg agttattagc 1920 ctctctggca ggtgggcaaa ccgaggcatg
gaggtttgtt taaggtgaac tgccagtgtg 1980 tgaccaccta gtggggtaga
gctgatgatt gcctcacacc ggagctcctt cctgtgccgc 2040 gttctgtcca
gaagacacag ccatggatgt ccattttagg atcagccaag ccccgtcttg 2100
tccttcattt ttattttatg tttttttaga aatggggtct tgctctgtca cccaggctgg
2160 gtgcagtggt gtgatcatag ctcaccgcag ctttgacgcc gtcttcccac
tcagtctact 2220 aagcttggac tataggccaa gactatagag tggtccttct
ttccattctt ttgggaccat 2280 gagaggccac ccatgtttcc tgcccctgct
gggccctgct gctcagaagg catggtctga 2340 ggctttcacc ttggtcgtga
gccttcgtgg tggtttcttt cagcatgggg ttgggatgct 2400 gtgctcaggc
ttctgcatgg tttcccacac tctcttctcc tcctcaggac tggatcatcg 2460
cgcctgaagg ctacgcgcgc tactactgtg agggggagtg tgccttccct ctgaactcct
2520 acatgaacgc caccaaccac gccatcgtgc agacgctggt gggtgtcacg
ccatcttggg 2580 gtgtggtcac ctgggccggg caggctgcgg ggccaccaga
tcctgctgcc tccaagctgg 2640 ggcctgagta gatgtcagcc cattgccatg
tcatgacttt tgggggcccc ttgcgccgtt 2700 aaaaaaaaat caaaaattgt
actttatgac tggtttggta taaagaggag tataatcttc 2760 gaccctggag
ttcatttatt tctcctaatt tttaaagtaa ctaaaagttg tatgggctcc 2820
tttgaggatg cttgtagtat tgtgggtgct ggttacggtg cctaagagca ctgggcccct
2880 gcttcatttt ccagtagagg aaacaggtaa acagatgaga aatttcagtg
aggggcacag 2940 tgatcagaag cgggccagca ggataatggg atggagagat
gagtggggac ccatgggcca 3000 tttcaagtta aatttcagtc gggtcaccag
gaagattcca tgtgataatg agattaacgt 3060 gcccagtcac ggcgacactc
agtaggtgtt attcctgctc tgccaacagc aaccatagtt 3120 gataagagct
gttagggatt ttgtcctttt gcttagaatc caaggttcaa ggaccttggt 3180
tatgtagctc cctgtcatga acatcatctg agcctttcct gcctactgat catccaccct
3240 gccttgaatg cttctagtga cagagagctc actaccagga ctactccctc
ctttcattta 3300 gtaatctgcc tccttctttt cttgtccctg tcctgtgtgt
taagtcctgg agaaaaatct 3360 catctatccc tttcatttga ttctgctctt
tgagggcagg ggtttttgtt tctttgtttg 3420 tttttttaag tgttggtttt
ccaaagccct tgctcccctc ctcaattgaa acttcaaagc 3480 cctcattggg
attgaaggtc cttaggctgg aaacagaaga gtcctcccca acctgttccc 3540
tggcctggat gtgctgtgct gtgccagtat cccctggaag gtgccaggca tgtctccccg
3600 gctgccaggg gacacatctc tatccttctc caacccctgc cttcatggcc
catggaacag 3660 gagtgccatc gccctgtgtg cacctacttc catcagtatt
tcaccagaga tctgcaggat 3720 caaagtgaat tctccaggga ttgtgaaatg
atgcgattgt ggtcatgttt aaaagggggc 3780 aactgtcttc tagagagtcc
tgatgaaatg cttccagagg aaatgagctg atggctggaa 3840 tttgctttaa
aatcattcaa ggtggagcag gtggggaagg gtatggatgt gtaagagttt 3900
gaaattgtcc atcataaaat gtgtaaaaag catgctggcc tatgtcagca gtcacagcct
3960 ggaggtggta acagagtgcc agtcactgat gctcaagcct ggcacctaca
gttgctggaa 4020 acccagaagt ttcacgttga aaacaacagg acagtggaat
ctctggccct gtcttgaaca 4080 cgtggcagat ctgctaacac tgatcttggt
tggctgccgt cagcttaggt tgagtggcgg 4140 tcttccctta gtttgcttag
tccccgctat tccctattgt cttacctcgg tctattttgc 4200 ttatcagtgg
acctcacgag gcactcatag gcatttgagt ctatgtgtcc ctgtcccaca 4260
tcctctgtaa ggtgcagaga agtccatgag caagatggag cacttctagt gggtccaagt
4320 cagggacact attcagcaat ctacagtgca cagggcagtt ccccaacaga
gaattacctg 4380 gtcctgaatg tcggatctgg ccccttcctt ccccactgta
taatgtgaaa acctctatgc 4440 tttgttcccc ttgtctgcaa aacagggata
atcccagaac tgagttgtcc atgtaaagtg 4500 cttagaacag ggagtgcttg
gcttggggag tgtcacctgc agtcattcat tatgcccaga 4560 caggatgttt
ctttatagaa acgtggaggc cagttagaac gactcaccgc ttctcaccac 4620
tgcccatgtt ttggtgtgtg tttcaggtcc acttcatcaa cccggaaacg gtgcccaagc
4680 cctgctgtgc gcccacgcag ctcaatgcca tctccgtcct ctacttcgat
gacagctcca 4740 acgtcatcct gaagaaatac agaaacatgg tggtccgggc
ctgtggctgc cactagctcc 4800 tccga 4805 41 314 DNA Artificial
Sequence consensus probe 41 gatcctaatg ggctgtacgt ggacttccag
cgcgacgtgg gctgggacga ctggatcatc 60 gcccccgtcg acttcgacgc
ctactactgc tccggagcct gccagttccc ctctgcggat 120 cacttcaaca
gcaccaacca cgccgtggtg cagaccctgg tgaacaacat gaaccccggc 180
aaggtaccca agccctgctg cgtgcccacc gagctgtccg ccatcagcat gctgtacctg
240 gacgagaatt ccaccgtggt gctgaagaac taccaggaga tgaccgtggt
gggctgcggc 300 tgccgctaac tgca 314 42 314 DNA Artificial Sequence
OP1 42 tgtaagaagc acgagctgta tgtcagcttc cgagacctgg gctggcagga
ctggatcatc 60 gcgcctgaag gctacgcgcg ctactactgt gagggggagt
gtgccttccc tctgaactcc 120 tacatgaacg ccaccaacca cgccatcgtg
cagacgctgg tccacttcat caacccggaa 180 acggtgccca agccctgctg
tgcgcccacg cagctcaatg ccatctccgt cctctacttc 240 gatgacagct
ccaacgtcat cctgaagaaa tacagaaaca tggtggtccg ggcctgtggc 300
tgccactagc tcct 314 43 315 DNA Artificial Sequence Figure 13 43 gat
cct aat ggg ctg tac gtg gac ttc cag cgc gac gtg ggc tgg gac 48 Asp
Pro Asn Gly Leu Tyr Val Asp Phe Gln Arg Asp Val Gly Trp Asp 1 5 10
15 gac tgg atc atc gcc ccc gtc gac ttc gac gcc tac tac tgc tcc gga
96 Asp Trp Ile Ile Ala Pro Val Asp Phe Asp Ala Tyr Tyr Cys Ser Gly
20 25 30 gcc tgc cag ttc ccc tct gcg gat cac ttc aac agc acc aac
cac gcc 144 Ala Cys Gln Phe Pro Ser Ala Asp His Phe Asn Ser Thr Asn
His Ala 35 40 45 gtg gtg cag acc ctg gtg aac aac atg aac ccc ggc
aag gta ccc aag 192 Val Val Gln Thr Leu Val Asn Asn Met Asn Pro Gly
Lys Val Pro Lys 50 55 60 ccc tgc tgc gtg ccc acc gag ctg tcc gcc
atc agc atg ctg tac ctg 240 Pro Cys Cys Val Pro Thr Glu Leu Ser Ala
Ile Ser Met Leu Tyr Leu 65 70 75 80 gac gag aat tcc acc gtg gtg ctg
aag aac tac cag gag atg acc gtg 288 Asp Glu Asn Ser Thr Val Val Leu
Lys Asn Tyr Gln Glu Met Thr Val 85 90 95 gtg ggc tgc ggc tgc cgc
taactgcag 315 Val Gly Cys Gly Cys Arg 100 44 102 PRT Artificial
Sequence Synthetic Construct 44 Asp Pro Asn Gly Leu Tyr Val Asp Phe
Gln Arg Asp Val Gly Trp Asp 1 5 10 15 Asp Trp Ile Ile Ala Pro Val
Asp Phe Asp Ala Tyr Tyr Cys Ser Gly 20 25 30 Ala Cys Gln Phe Pro
Ser Ala Asp His Phe Asn Ser Thr Asn His Ala 35 40 45 Val Val Gln
Thr Leu Val Asn Asn Met Asn Pro Gly Lys Val Pro Lys 50 55 60 Pro
Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu 65 70
75 80 Asp Glu Asn Ser Thr Val Val Leu Lys Asn Tyr Gln Glu Met Thr
Val 85 90 95 Val Gly Cys Gly Cys Arg 100 45 4 PRT Artificial
Sequence Hinge region 45 Asp Pro Asn Gly 1 46 106 PRT Artificial
Sequence beta-inhibin-a 46 Cys Cys Lys Lys Gln Phe Phe Val Ser Phe
Lys Asp Ile Gly Trp Asn 1 5 10 15 Asp Trp Ile Ile Ala Pro Ser Gly
Tyr His Ala Asn Tyr Cys Glu Gly 20 25 30 Glu Cys Pro Ser His Ile
Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe 35 40 45 His Ser Thr Val
Ile Asn His Tyr Arg Met Arg Gly His Ser Pro Phe 50 55 60 Ala Asn
Leu Lys Ser Cys Cys Val Pro Thr Lys Leu Arg Pro Met Ser 65 70 75 80
Met Leu Tyr Tyr Asp Asp Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln 85
90 95 Asn Met Ile Val Glu Glu Cys Gly Cys Ser 100 105 47 105 PRT
Artificial Sequence beta-inhibin-b 47 Cys Cys Arg Gln Gln Phe Phe
Ile Asp Phe Arg Ile Gly Trp Asn Asp 1 5 10 15 Trp Ile Ile Ala Pro
Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser 20 25 30 Cys Pro Ala
Tyr Leu Ala Gly Val Pro Gly Ser Ala Ser Ser Phe His 35 40 45 Thr
Ala Val Val Asn Gln Tyr Arg Met Arg Gly Leu Asn Pro Gly Thr 50 55
60 Lys Val Asn Ser Cys Cys Ile Pro Thr Lys Leu Ser Thr Met Ser Met
65 70 75 80 Leu Tyr Phe Asp Asp Glu Tyr Asn Ile Val Lys Arg Asp Val
Pro Asn 85 90 95 Met Ile Val Glu Glu Cys Gly Cys Ala 100 105 48 99
PRT Artificial Sequence TGF-beta-1 48 Cys Cys Val Arg Gln Leu Tyr
Ile Asp Phe Arg Lys Asp Leu Gly Trp 1 5 10 15 Lys Trp Ile His Glu
Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly 20 25 30 Pro Cys Pro
Tyr Ile Trp Ser Leu Leu Asp Thr Gln Tyr Ser Lys Val 35 40 45 Leu
Ala Leu Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys 50 55
60 Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly
65 70 75 80 Arg Lys Pro Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg
Ser Cys 85 90 95 Lys Cys Ser 49 99 PRT Artificial Sequence
TGF-beta-2 49 Cys Cys Leu Arg Pro Leu Tyr Ile Asp Phe Lys Arg Asp
Leu Gly Trp 1 5 10 15 Lys Trp Ile His Glu Pro Lys Gly Tyr Asn Ala
Asn Phe Cys Ala Gly 20 25 30 Ala Cys Pro Tyr Leu Trp Ser Leu Ser
Asp Thr Gln His Ser Arg Val 35 40 45 Leu Ser Leu Tyr Asn Thr Ile
Asn Pro Glu Ala Ser Ala Ser Pro Cys 50 55 60 Cys Val Ser Gln Asp
Leu Glu Pro Leu Thr Ile Leu Tyr Tyr Ile Gly 65 70 75 80 Lys Thr Pro
Lys Ile Glu Gln Leu Ser Asn Met Ile Val Lys Ser Cys 85 90 95 Lys
Cys Ser 50 99 PRT Artificial Sequence TGF-beta-3 50 Cys Cys Val Arg
Pro Leu Tyr Ile Asp Phe Arg Gln Asp Leu Gly Trp 1 5 10 15 Lys Trp
Val His Glu Pro Lys Gly Tyr Tyr Ala Asn Phe Cys Ser Gly 20 25 30
Pro Cys Pro Tyr Leu Arg Ser Leu Ala Asp Thr Thr His Ser Thr Val 35
40 45 Leu Gly Leu Tyr Asn Thr Leu Asn Pro Glu Ala Ser Ala Ser Pro
Cys 50 55 60 Cys Val Pro Gln Asp Leu Glu Pro Leu Thr Ile Leu Tyr
Tyr Val Gly 65 70 75 80 Arg Thr Pro Lys Val Glu Gln Leu Ser Asn Met
Val Val Lys Ser Cys 85 90 95 Lys Cys Ser 51 99 PRT Artificial
Sequence MIS 51 Cys Ala Leu Arg Glu Leu Ser Val Asp Leu Arg Ala Glu
Arg Ser Val 1 5 10 15 Leu Ile Pro Glu Thr Tyr Gln Ala Asn Asn Cys
Gln Gly Val Cys Gly 20 25 30 Trp Pro Gln Ser Asp Arg Asn Pro Arg
Tyr Gly Asn His Val Val Leu 35 40 45 Leu Leu Lys Met Gln Ala Arg
Gly Ala Ala Leu Ala Arg Pro Pro Cys 50 55 60 Cys Val Pro Thr Ala
Tyr Ala Gly Lys Leu Leu Ile Ser Leu Ser Glu 65 70 75 80 Glu Arg Ile
Ser Ala His His Val Pro Asn Met Val Ala Thr Glu Cys 85 90 95 Gly
Cys Arg 52 103 PRT Artificial Sequence Alpha-inhibin 52 Cys His Arg
Val Ala Leu Asn Ile Ser Phe Gln Glu Leu Gly Trp Glu 1 5 10 15 Arg
Trp Ile Val Tyr Pro Pro Ser Phe Ile Phe His Tyr Cys His Gly 20 25
30 Gly Cys Gly Leu His Ile Pro Pro Asn Leu Ser Leu Pro Val Pro Gly
35 40 45 Ala Pro Pro Thr Pro Ala Gln Pro Tyr Ser Leu Leu Pro Gly
Ala Gln 50 55 60 Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg Pro
Leu His Val Arg 65 70 75 80 Thr Thr Ser Asp Gly Gly Tyr Ser Phe Lys
Tyr Glu Xaa Asn Leu Leu 85 90 95 Thr Gln His Cys Ala Cys Ile 100 53
861 DNA Artificial Sequence COP-5 fusion protein 53 atg aaa gca att
ttc gta ctg aaa ggt tca ctg gac aga gat ctg gac 48 Met Lys Ala Ile
Phe Val Leu Lys Gly Ser Leu Asp Arg Asp Leu Asp 1 5 10 15 tct cgt
ctg gat ctg gac gtt cgt acc gac cac aaa gac ctg tct gat 96 Ser Arg
Leu Asp Leu Asp Val Arg Thr Asp His Lys Asp Leu Ser Asp 20 25 30
cac ctg gtt ctg gtc gac ctg gct cgt aac gac ctg gct cgt atc gtt 144
His Leu Val Leu Val Asp Leu Ala Arg Asn Asp Leu Ala Arg Ile Val 35
40 45 act ccc ggg tct cgt tac gtt gcg gat ctg gaa ttc atg gct gac
aac 192 Thr Pro Gly Ser Arg Tyr Val Ala Asp Leu Glu Phe Met Ala Asp
Asn 50 55 60 aaa ttc aac aag gaa cag cag aac gcg ttc tac gag atc
ttg cac ctg 240 Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile
Leu His Leu 65 70 75 80 ccg aac ctg aac gaa gag cag cgt aac ggc ttc
atc caa agc ttg aag 288 Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe
Ile Gln Ser Leu Lys 85 90 95 gat gag ccc tct cag tct gcg aat ctg
cta gcg gat gcc aag aaa ctg 336 Asp Glu Pro Ser Gln Ser Ala Asn Leu
Leu Ala Asp Ala Lys Lys Leu 100 105 110 aac gat gcg cag gca ccg aaa
tcg gat cag ggg caa ttc atg gct gac 384 Asn Asp Ala Gln Ala Pro Lys
Ser Asp Gln Gly Gln Phe Met Ala Asp 115 120 125 aac aaa ttc aac aag
gaa cag cag aac gcg ttc tac gag atc ttg cac 432 Asn Lys Phe Asn Lys
Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His 130 135 140 ctg ccg aac
ctg aac gaa gag cag cgt aac ggc ttc atc caa agc ttg 480 Leu Pro Asn
Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu 145 150 155 160
aag gat gag ccc tct cag tct gcg aat ctg cta gcg gat gcc aag aaa 528
Lys Asp Glu Pro Ser Gln Ser Ala Asn Leu Leu Ala Asp Ala Lys Lys 165
170 175 ctg aac gat gcg cag gca ccg aag gat cct aat ggg ctg tac gtc
gac 576 Leu Asn Asp Ala Gln Ala Pro Lys Asp Pro Asn Gly Leu Tyr Val
Asp 180 185 190 ttc agc gac gtg ggc tgg gac gac tgg att gtg gcc cca
cca ggc tac 624 Phe Ser Asp Val Gly Trp Asp Asp Trp Ile Val Ala Pro
Pro Gly Tyr 195 200 205 cag gcc ttc tac tgc cat ggc gaa tgc cct ttc
ccg cta gcg gat cac 672 Gln Ala Phe Tyr Cys His Gly Glu Cys Pro Phe
Pro Leu Ala Asp His 210 215 220 ttc aac agc acc aac cac gcc gtg gtg
cag acc ctg gtg aac tct gtc 720 Phe Asn Ser Thr Asn His Ala Val Val
Gln Thr Leu Val Asn Ser Val 225 230 235 240 aac tcc aag atc cct aag
gct tgc tgc gtg ccc acc gag ctg tcc gcc 768 Asn Ser Lys Ile Pro Lys
Ala Cys Cys Val Pro Thr Glu Leu Ser Ala 245 250 255 atc agc atg ctg
tac ctg gac gag aat gag aag gtg gtg ctg aag aac 816 Ile Ser Met Leu
Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn 260 265 270 tac cag
gag atg gta gta gag ggc tgc ggc tgc cgc taactgcag 861 Tyr Gln Glu
Met Val Val Glu Gly Cys Gly Cys Arg 275 280 54 284 PRT Artificial
Sequence Synthetic Construct 54 Met Lys Ala Ile Phe Val Leu Lys Gly
Ser Leu Asp Arg Asp Leu Asp 1 5 10 15 Ser Arg Leu Asp Leu Asp Val
Arg Thr Asp His Lys Asp Leu Ser Asp 20 25 30 His Leu Val Leu Val
Asp Leu Ala Arg Asn Asp Leu Ala Arg Ile Val 35 40 45 Thr Pro Gly
Ser Arg Tyr Val Ala Asp Leu Glu Phe Met Ala Asp Asn 50 55 60 Lys
Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 65 70
75 80 Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu
Lys 85 90 95 Asp Glu Pro Ser Gln Ser Ala Asn Leu Leu Ala Asp Ala
Lys Lys Leu 100 105 110 Asn Asp Ala Gln Ala Pro Lys Ser Asp Gln Gly
Gln Phe Met Ala Asp 115 120 125 Asn Lys Phe Asn Lys Glu Gln Gln Asn
Ala Phe Tyr Glu Ile Leu His 130 135 140 Leu Pro Asn Leu Asn Glu Glu
Gln Arg Asn Gly Phe Ile Gln Ser Leu
145 150 155 160 Lys Asp Glu Pro Ser Gln Ser Ala Asn Leu Leu Ala Asp
Ala Lys Lys 165 170 175 Leu Asn Asp Ala Gln Ala Pro Lys Asp Pro Asn
Gly Leu Tyr Val Asp 180 185 190 Phe Ser Asp Val Gly Trp Asp Asp Trp
Ile Val Ala Pro Pro Gly Tyr 195 200 205 Gln Ala Phe Tyr Cys His Gly
Glu Cys Pro Phe Pro Leu Ala Asp His 210 215 220 Phe Asn Ser Thr Asn
His Ala Val Val Gln Thr Leu Val Asn Ser Val 225 230 235 240 Asn Ser
Lys Ile Pro Lys Ala Cys Cys Val Pro Thr Glu Leu Ser Ala 245 250 255
Ile Ser Met Leu Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys Asn 260
265 270 Tyr Gln Glu Met Val Val Glu Gly Cys Gly Cys Arg 275 280 55
15 PRT Artificial Sequence BOP 55 Ser Phe Asp Ala Tyr Tyr Cys Ser
Gly Ala Cys Gln Phe Pro Ser 1 5 10 15 56 15 PRT Artificial Sequence
DPP 56 Gly Tyr Asp Ala Tyr Tyr Cys His Gly Lys Cys Pro Phe Phe Leu
1 5 10 15 57 15 PRT Artificial Sequence Vg1 57 Gly Tyr Met Ala Asn
Tyr Cys Tyr Gly Glu Cys Pro Tyr Pro Leu 1 5 10 15 58 15 PRT
Artificial Sequence inhibin 58 Gly Tyr His Ala Asn Tyr Cys Glu Gly
Glu Cys Pro Ser His Ile 1 5 10 15 59 15 PRT Artificial Sequence
TGF-beta 59 Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys Pro Tyr Ile
Trp 1 5 10 15 60 21 PRT Artificial Sequence BOP 60 Lys Arg Ala Cys
Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu 1 5 10 15 Tyr Leu
Asp Glu Asn 20 61 20 PRT Artificial Sequence Vg1 61 Leu Pro Cys Cys
Val Pro Thr Lys Met Ser Pro Ile Ser Met Leu Phe 1 5 10 15 Tyr Asp
Asn Asn 20 62 20 PRT Artificial Sequence inhibin 62 Lys Ser Cys Cys
Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr 1 5 10 15 Tyr Asp
Asp Gly 20 63 19 PRT Artificial Sequence TGF-beta 63 Ala Pro Cys
Cys Val Pro Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr 1 5 10 15 Tyr
Val Gly 64 20 PRT Artificial Sequence DPP 64 Lys Ala Cys Cys Val
Pro Thr Gln Leu Asp Ser Val Ala Met Leu Tyr 1 5 10 15 Leu Asn Asp
Gln 20 65 5 PRT Artificial Sequence BOP and DPP match sequence 65
Leu Tyr Val Asp Phe 1 5 66 5 PRT Artificial Sequence Vg1 66 Leu Tyr
Val Asp Phe 1 5 67 5 PRT Artificial Sequence Vgl 67 Leu Tyr Val Glu
Phe 1 5 68 5 PRT Artificial Sequence TGF-beta 68 Leu Tyr Ile Asp
Phe 1 5 69 5 PRT Artificial Sequence inhibin 69 Phe Phe Val Ser Phe
1 5 70 5 PRT Artificial Sequence N-terminal sequence 70 Cys Lys Arg
His Pro 1 5 71 5 PRT Artificial Sequence N-terminal sequence 71 Cys
Arg Arg Lys Gln 1 5 72 5 PRT Artificial Sequence N-terminal
sequence 72 Cys Lys Arg His Glu 1 5
* * * * *