U.S. patent number RE43,166 [Application Number 12/613,119] was granted by the patent office on 2012-02-07 for gene encoding chondroitinase abc and uses therefor.
This patent grant is currently assigned to Maruha Nichiro Foods, Inc.. Invention is credited to Hiroshi Oda, Nobuyuki Sato, Masahiko Shimada.
United States Patent |
RE43,166 |
Sato , et al. |
February 7, 2012 |
Gene encoding chondroitinase ABC and uses therefor
Abstract
Nucleic acid sequences coding for the chondroitinase ABC gene
and isolated chondroitinase ABE protein produced in a host cell
transformed with a nucleic acid vector directing the expression of
a nucleotide sequence coding for chondroitinase ABE protein
described. Chondroitinase ABC prepared by chemical synthesis also
described. Monoclonal and polyclonal antibodies which are
specifically reactive with chondroitinase ABC protein are
disclosed. The isolated chondroitinase ABC can be used in methods
of treating intervertebral disc replacement, promoting neurite
regeneration, and detecting galactosaminoglycans.
Inventors: |
Sato; Nobuyuki (Ibaraki,
JP), Shimada; Masahiko (Ibaraki, JP), Oda;
Hiroshi (Ibaraki, JP) |
Assignee: |
Maruha Nichiro Foods, Inc.
(Tokyo, JP)
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Family
ID: |
35966205 |
Appl.
No.: |
12/613,119 |
Filed: |
November 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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08184435 |
Jan 14, 1994 |
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08074349 |
Jun 8, 1993 |
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Reissue of: |
08488960 |
Jun 7, 1995 |
7008783 |
Mar 7, 2006 |
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Foreign Application Priority Data
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Sep 22, 1992 [JP] |
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4-253016 |
Feb 24, 1993 [JP] |
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5-35810 |
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Current U.S.
Class: |
435/232;
536/23.2; 435/252.33; 435/69.1; 435/320.1; 435/254.11; 435/252.3;
435/325 |
Current CPC
Class: |
C12Y
402/02004 (20130101); C12N 9/88 (20130101) |
Current International
Class: |
C12N
15/60 (20060101); C12N 5/10 (20060101); C12N
1/00 (20060101); C12N 1/21 (20060101); C12N
15/63 (20060101); C12N 9/88 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 355 831 |
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Feb 1990 |
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EP |
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0355831 |
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Feb 1990 |
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EP |
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0-576-294 |
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Dec 1993 |
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EP |
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0576294 |
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Dec 1993 |
|
EP |
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91/06303 |
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May 1991 |
|
WO |
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91/16070 |
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Oct 1991 |
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WO |
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WO 91/16070 |
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Oct 1991 |
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WO |
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by other.
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Primary Examiner: Prouty; Rebecca
Attorney, Agent or Firm: Nelson Mullins Riley &
Scarborough LLP Remillard, Esq.; Jane E. Sloper, Esq.; Jill
Gorny
Parent Case Text
This application is a continuation of application Ser. No.
08/184,435 filed on Jan. 14, 1994 now abandoned Entitled: Gene
Encoding Chondroitinase ABC And Uses Therefor, which is a
divisional of Ser. No. 08/074,349 filed Jun. 8, 1993 now abandoned.
Claims
What is claimed is:
.[.1. An isolated nucleic acid fragment encoding chondroitinase
ABC, comprising the nucleotide sequence of SEQ ID NO: 1..].
.[.2. An expression vector comprising the nucleic acid as defined
in claim 1 operably linked to a regulatory sequence..].
.[.3. A host cell transformed with the expression vector as defined
in claim 2..].
4. A host cell of claim .[.3.]. .Iadd.23 .Iaddend.wherein the cell
is eukaryotic.
5. A host cell of claim .[.3.]. .Iadd.23 .Iaddend.wherein the cell
is prokaryotic.
.[.6. A method of producing chondroitinase ABC protein comprising:
culturing the host cell as defined in claim 2 under conditions
appropriate for expression; and isolating chondroitinase ABC
protein from the culture..].
.[.7. An isolated nucleic acid encoding chondroitinase ABC
comprising a nucleotide sequence which differs from the nucleotide
sequence of SEQ ID NO: 1, due to degeneracy in the genetic
code..].
.[.8. An expression vector comprising the nucleic acid as defined
in claim 7 operably linked to a regulatory sequence..].
.[.9. A host cell transformed with the expression vector as defined
in claim 8..].
.[.10. A method of producing chondroitinase ABC protein comprising:
culturing the host cell as in defined in claim 9 under conditions
appropriate for expression; and isolating chondroitinase ABC
protein from the culture..].
11. An isolated nucleic acid fragment comprising .[.the coding
region of chondroitinase ABC and having a nucleotide sequence
consisting of.]. nucleotides .[.297-3288.]. .Iadd.73 to 3066
.Iaddend.of SEQ ID NO: 1.
12. An expression vector comprising the nucleic acid of claim 11
operably linked to a regulatory sequence.
13. A host cell transformed with the expression vector of claim
12.
14. A method of producing chondroitinase ABC protein comprising:
culturing the host cell as in defined in claim 13 under conditions
appropriate for expression; and isolating chondroitinase ABC
protein from the culture.
15. An isolated nucleic acid comprising a nucleotide sequence which
differs from nucleotides .[.297-3288.]. .Iadd.73 to 3066
.Iaddend.of SEQ ID NO: 1, due to degeneracy in the genetic
code.
16. An expression vector comprising the nucleic acid as defined in
claim 15 operably linked to a regulatory sequence.
.[.17. An isolated nucleic acid fragment comprising nucleotides
2160-3288 of SEQ ID NO: 1..].
.[.18. An expression vector comprising the nucleic acid as defined
in claim 17 operably linked to a regulatory sequence..].
.[.19. An isolated nucleic acid comprising a nucleotide sequence
which differs from nucleotides 2160-3288 of SEQ ID NO: 1, due to
degeneracy in the genetic code..].
.[.20. An expression vector comprising the nucleic acid as defined
in claims operably linked to a regulatory sequence..].
21. An isolated nucleic acid fragment comprising the coding region
of the nucleotide sequence of SEQ ID NO: 1.
22. An expression vector comprising the nucleic acid as defined in
claim 21 operably linked to a regulatory sequence.
23. A host cell transformed with the expression vector as defined
in claim 22.
24. A method of producing chondroitinase ABC protein comprising:
culturing the host cell as in defined in claim 23 under conditions
appropriate for expression; and isolating chondroitinase ABC
protein from the culture.
25. An isolated nucleic acid comprising a nucleotide sequence which
differs from the coding region of SEQ ID NO: 1, due to degeneracy
in the genetic code.
26. An expression vector comprising the nucleic acid as defined in
claim 25 operably linked to a regulatory sequence.
27. A host cell transformed with the expression vector as defined
in claim 26.
28. A method of producing chondroitinase ABC protein comprising:
culturing the host cell as in defined in claim 27 under conditions
appropriate for expression; and isolating chondroitinase ABC
protein from the culture.
.Iadd.29. An isolated nucleic acid fragment encoding chondroitinase
ABC, wherein the nucleic acid comprises a nucleotide sequence of
the insert of pCHS6 obtained from E. coli XL1-Blue/pCHS6 deposited
at Accession NO. FERM BP-4170 ..Iaddend.
Description
BACKGROUND OF THE INVENTION
Chondroitin lyase (EC 4.2.2.4) or chondroitinase ABC is an enzyme
which catalyzes the depolymerization of chondroitin sulfate.
Through .beta.-elimination of 1,4 hexosaminidic bonds,
chondroitinase ABC degrades chondroitin, chondroitin 4-sulfate
(chondroitin A sulfate), dermatan sulfate (chondroitin B sulfate),
chondroitin 6-sulfate (chondroitin C sulfate) and hyaluronate to
the respective unsaturated disaccharides (.DELTA.di-OS for
chondroitin, .DELTA.di-4S for chondroitin A sulfate, .DELTA.di-4-6S
for chondroitin B sulfate and .DELTA.di-6S for chondroitin C
sulfate, respectively). The enzyme has been isolated in various
strains of bacteria (Neuberg, C. et al., (1914) Biochem. Z. 67:
82-89) (Neuberg, C. et al. (1931) Biochem, Z. 234: 345-346;
Yamagata, T. et al., (1968) J. Biol. Chem. 243: 1523-1535)
including Proteus vulgaris (Yamagata, T. et al. (1968) J. Biol.
Chem. 243: 1523-1535; Thurston, C. F. (1974) J. Gen. Microbiol. 80:
515-522; Sato N. et al. (1986) Agric. Biol. Chem. 50: 1057-1059;
Sato N. et al. (1986) Biotechnol. Bioeng. 28: 1707-1712; Sato, N.
et al. (1986) J. Ferment. Technol. 64: 155-159).
Chondroitin sulfate consists of alternating .beta. 1-3 glucuronidic
and .beta. 1-4 N-acetylgalactosaminidic bonds, and is sulfated at
either C-4 or C-6 of the N-acetylgalactosamine pyranose.
Chondroitin sulfate is known to be widely distributed in mammalian
tissue, such as in skin, cornea, bone and especially in cartilage.
Thus, chondroitinase ABC has been used as an experimental reagent
for the determination or quantitation of total amount of
galactosaminoglycans in the field of orthopedic surgery (Linker, A.
et al. (1960) J. Biol. Chem. 235: 3061-3065; Saito, H. et al.
(1968) J. Biol. Chem. 243: 1536-1542; Pettipher, E. R. et al.
(1989) Arthritis Rheum. 32: 601-607; Caterson, B. et al. (1990) J.
Cell Science 97: 411-417; and Seibel, M. J. et al. (1992) Arch.
Biochem. Biophys. 296: 410-418).
Recently, chondroitinase ABC has been reported to be a potential
reagent for chemonucleolysis, an established treatment for
intervertebral disc displacement (Kato, F. et al. (1990) Clin.
Orthop. 253: 301-308; Henderson, N. et al. (1991) Spine 16:
203-209). However, for the utilization of chondroitinase ABC as a
clinical reagent, there are many problems to be overcome. For
example, the preparation of chondroitinase ABC from P. vulgaris
requires tedious and intricate procedures, since the cellular
content of the enzyme is low. Therefore, an efficient method for
the efficient preparation of highly purified chondroitinase ABC is
now sought.
SUMMARY OF THE INVENTION
This invention pertains to nucleic acid sequences coding for the
chondroitinase ABC gene and isolated chondroitinase ABC protein
produced in a host cell transformed with a nucleic acid vector
directing the expression of a nucleotide sequence coding for
chondroitinase ABC. Chondroitinase ABC prepared by chemical
synthesis is also provided. This invention further provides
monoclonal and polyclonal antibodies which are specifically
reactive with chondroitinase ABC. The isolated chondroitinase ABC
can be used in methods of treating intervertebral disc displacement
and promoting neurite regeneration or in method of detecting the
presence of galactosaminoglycans.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1-A and 1-B show the primers used for polymerase chain
reaction (PCR) amplification of chondroitinase ABC from P. vulgaris
genomic DNA.[.;.]..Iadd.. FIG. 1A provides the amino acid sequence
of the N-terminal region of purified chondroitinase ABC (SEQ ID NO:
3), the nucleotide sequences of the sense primers (SEQ ID NOS 5 and
6), and the nucleotide sequences of the antisense primers (SEQ ID
NOS 7 and 8)..Iaddend.
FIG. 1-B also shows the probe used for plaque
hybridization.[.;.]..Iadd.. FIG. 1B provides the amino acid
sequence of the N-terminal region of purified chondroitinase ABC
(SEQ ID NO: 3), the nucleotide sequence of primer A (SEQ ID NO:
10), the nucleotide sequence of the probe (SEQ ID NO: 9), the
nucleotide sequence of primer B (SEQ ID NO: 11), and the full
length DNA sequences (SEQ ID NOS 12 and 13)..Iaddend.
FIG. 1-C shows the restriction maps for three recombinant phages
and the fragment of phage 11-5 which was subcloned into pSTV29 for
sequencing.
FIG. 2 shows the construction of pCHSP, a hybrid plasmid containing
the putative promoter region of chondroitinase ABC (SEQ ID NO:
14).
FIG. 3 shows primer extension analysis using a sequencing ladder
(SEQ ID NO:15).
FIG. 4 shows the nucleotide sequence of the promoter region of
chondroitinase ABC (SEQ ID NO: 16) and the peptide sequence (SEQ ID
NO:17).
FIG. 5 shows the construction of plasmids pCHS 6, pCHS.DELTA. 6,
and pCHS 26 each of which contains a fragment of the chondroitinase
ABC gene.
FIG. 6 shows SDS-PAGE and immunoblot analysis of recombinant
chondroitinase ABC protein produced by pCHS.DELTA. 6 transformed E.
coli (lane 1); protein produced by pSTV 29 without the
chondroitinase ABC gene in E. coli (lane 2); natural chondroitinase
ABC produced by P. vulgaris (lane 3); and molecular weight markers
(lane 4).
FIG. 7 shows the DNA (SEQ ID NO:1) and amino acid sequence (SEQ ID
NO:2) of the chondroitinase ABC gene.[.including non-coding
regions.]..
DETAILED DESCRIPTION OF THE INVENTION
This invention pertains to nucleic acid sequences coding for
chondroitinase ABC, an enzyme which degrades chondroitin A, B, and
C. The chondroitinase ABC gene was derived using recombinant DNA
techniques. A nucleic acid sequence coding for chondroitinase ABC
preferably has the sequence shown in SEQ ID NO: 1 (FIG. 7). The
deduced amino acid sequence of chondroitinase ABC is shown in SEQ
ID NO:2 (FIG. 7).
Accordingly, one aspect of the invention pertains to an isolated
nucleic acid having a nucleotide sequence coding for chondroitinase
ABC, fragments thereof, or equivalents thereof. The term nucleic
acid as used herein is intended to include such fragments or
equivalents. A nucleic acid sequence coding for chondroitinase ABC
can obtained from mRNA present in Proteus vulgaris. Nucleic acid
sequences coding for chondroitinase ABC can also be obtained from
P. vulgaris genomic DNA. The nucleic acid sequence coding for
chondroitinase ABC can be obtained using the method disclosed
herein or any other suitable technique for isolation and molecular
cloning of genes. The nucleic acid sequences of the invention can
be DNA or RNA. The preferred nucleic acid is a DNA having the
sequence depicted in SEQ ID NO:1 (FIG. 7) or equivalents
thereof.
The term equivalent is intended to include nucleotide sequences
coding for functionally equivalent chondroitinase ABC proteins. For
example, DNA sequence polymorphisms within the nucleotide sequence
of chondroitinase ABC (especially those within the third base of a
codon) may result in "silent" mutations which do not affect the
amino acid sequence of the chondroitinase ABC protein. However, it
is expected that DNA sequence polymorphisms that do lead to changes
in the amino acid sequence of chondroitinase ABC will exist. It
will be appreciated by one skilled in the art that these variations
in one or more nucleotides (up to about 3-4% of the nucleotides) of
the nucleic acid sequence coding for chondroitinase ABC may exist
due to natural allelic variation. Any and all such nucleotide
variations and resulting amino acid polymorphisms are within the
scope of the invention. Furthermore, there may be one or more
isoforms or related, cross-reacting family members of
chondroitinase ABC. Such isoforms or family members are defined as
proteins related in function and amino acid sequence to
chondroitinase ABC, but encoded by genes at different loci.
A fragment of the nucleic acid sequence coding for chondroitinase
ABC is defined as a nucleotide sequence having fewer nucleotides
than the nucleotide sequence coding for the entire amino acid
sequence of chondroitinase ABC protein. Such fragments encode a
catalytically-active fragment of chondroitinase ABC protein which
depolymerizes chondroitin A, B, or C. Nucleic acid fragments within
the scope of the invention include those capable of hybridizing
with nucleic acid from other animal species for use in screening
protocols to detect chondroitinase ABC or enzymes that are
cross-reactive with chondroitinase ABC. Nucleic acid sequences
within the scope of the invention may also contain linker
sequences, modified restriction endonuclease sites and other
sequences useful for molecular cloning, expression or purification
of recombinant chondroitinase ABC or catalytically-active fragments
thereof.
This invention also provides expression vectors containing a
nucleic acid sequence coding for chondroitinase ABC, operably
linked to at least one regulatory sequence. Operably linked is
intended to mean that the nucleotide sequence is linked to a
regulatory sequence in a manner which allows expression of the
nucleotide sequence. Regulatory sequences are art-recognized and
are selected to direct expression of chondroitinase ABC.
Accordingly, the term regulatory sequence includes promoters,
enhancers and other expression control elements. Such regulatory
sequences are described in Goeddel, Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It should be understood that the design of the expression
vector may depend on such factors as the choice of the host cell to
be transformed and/or the type of protein desired to be
expressed.
This invention further pertains to a host cell transformed to
express chondroitinase ABC. The host cell may be any prokaryotic or
eukaryotic cell. For example, chondroitinase ABC protein may be
expressed in bacterial cells such as E. coli, insect cells
(baculovirus), yeast, or mammalian cells such as Chinese hamster
ovary cells (CHO). Other suitable host cells may be found in
Goeddel, (1990) supra or one known to those skilled in the art.
Expression in eukaryotic cells such as mammalian, yeast, or insect
cells can lead to partial or complete glycosylation and/or
formation of relevant inter- or intra-chain disulfide bonds of
recombinant protein. Examples of vectors for expression in yeast S.
cerivisae include pYepSec1 (Baldari. et al., (1987) EMBO J.
6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943),
pJRY88 (Schultz et al., (1987) Gene 54:113-123), and pYES2
(Invitrogen Corporation, San Diego, Calif.). Baculovirus vectors
available for expression of proteins in cultured insect cells (SF 9
cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol.
3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M.
D., (1989) Virology 170:31-39). Generally COS cells (Gluzman, Y.,
(1981) Cell 23:175-182) are used in conjunction with such vectors
as pCDM 8 (Aruffo, A. and Seed, B., (1987) Proc. Natl. Acad. Sci.
USA 84:8573-8577) for transient amplification/expression in
mammalian cells, while CHO (dhfr.sup.- Chinese Hamster Ovary) cells
are used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO
J. 6:187-195) for stable amplification/expression in mammalian
cells. Vector DNA can be introduced into mammalian cells via
conventional techniques such as calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, or
electroporation. Suitable methods for transforming host cells can
be found in Sambrook et al, Molecular Cloning: A Laboratory Manual,
2nd Edition, Cold Spring Harbor Laboratory press (1989), and other
laboratory textbooks.
Expression in prokaryotes is most often carried out in E. coli with
either fusion or non-fusion inducible expression vectors. Fusion
vectors usually add a number of NH.sub.2 terminal amino acids to
the expressed target gene. These NH.sub.2 terminal amino acids
often are referred to as a reporter group. Such reporter groups
usually serve two purposes: 1) to increase the solubility of the
target recombinant protein; and 2) to aid in the purification of
the target recombinant protein by acting as a ligand in affinity
purification. Often, in fusion expression vectors, a proteolytic
cleavage site is introduced at the junction of the reporter group
and the target recombinant protein to enable separation of the
target recombinant protein from the reporter group subsequent to
purification of the fusion protein. Such enzymes include Factor Xa,
thrombin and enterokinase. Typical fusion expression vectors
include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England
Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)
which fuse glutathione S-tranferase, maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
Inducible non-fusion expression vectors include pTrc (Amann et al.,
(1988) Gene 69:301-315) and pET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990) 60-89). While target gene expression
relies on host RNA polymerase transcription from the hybrid trp-lac
fusion promoter in pTrc, expression of target genes inserted into
pET 11d relies on transcription from the T7 gn10-lac 0 fusion
promoter mediated by coexpressed viral RNA polymerase (T7 gn1).
This viral polymerase is supplied by host strains BL21(DE3) or
HMS174(DE3) from a resident g prophage harboring a T7 gn1 under the
transcriptional control of the lacUV 5 promoter.
One strategy to maximize recombinant chondroitinase ABC expression
in E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, S., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 119-128). Another
strategy would be to alter the nucleic acid sequence of the
chondroitinase ABC gene to be inserted into an expression vector so
that the individual codons for each amino acid would be those
preferentially utilized in highly expressed E. coli proteins (Wada
et al., (1992) Nuc. Acids Res. 20:2111-2118). Such alteration of
nucleic acid sequences of the invention can be carried out by
standard DNA synthesis techniques.
The nucleic acid sequences of the invention can also be chemically
synthesized using standard techniques. Various methods of
chemically synthesizing polydeoxynucleotides are known, including
solid-phase synthesis which, like peptide synthesis, has been fully
automated in commercially available DNA synthesizers (See e.g.,
Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. U.S. Pat.
No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 and 4,373,071,
incorporated by reference herein).
This invention further pertains to methods of producing
chondroitinase ABC protein. For example, a host cell transformed
with a nucleic acid vector directing expression of a nucleotide
sequence coding for chondroitinase ABC protein can be cultured
under appropriate conditions to allow expression of chondroitinase
ABC to occur. The protein may be secreted and isolated from a
mixture of cells and medium containing chondroitinase ABC protein.
Alternatively, the protein may be retained cytoplasmically and the
cells harvested, lysed and the protein isolated. The culture
includes host cells, media and other byproducts. Suitable mediums
for cell culture are well known in the art. Chondroitinase ABC
protein can be isolated from cell culture medium, host cells, or
both using techniques known in the art for purifying proteins
including ion-exchange chromatography, gel filtration
chromatography, ultrafiltration, electrophoresis, and
immunoaffinity purification with antibodies specific for
chondroitinase ABC or fragment thereof.
Another aspect of the invention pertains to isolated chondroitinase
ABC protein. The term "chondroitinase ABC" or "chondroitinase ABC
protein" is intended to include functional equivalents thereof and
catalytically-active fragments thereof. The term functional
equivalent is intended to include proteins which differ in amino
acid sequence from the chondroitinase ABC sequence depicted in SEQ
ID NO:2 (FIG. 7) but where such differences result in a modified
protein which functions in the same or similar manner as
chondroitinase ABC or which has the same or similar characteristics
of chondroitinase ABC. For example, a functional equivalent of
chondroitinase ABC may have a modification such as a substitution,
addition or deletion of an amino acid residue which is not directly
involved in the enzyme activity of chondroitinase ABC (i.e., the
ability of chondroitinase ABC to depolymerize chondroitin
4-sulphate, chondroitin 6-sulfate, and dermatan sulfate). Various
modifications of the chondroitinase ABC protein to produce
functional equivalents of chondroitinase ABC are described in
detail herein.
The term isolated as used herein refers to chondroitinase ABC
protein substantially free of cellular material or culture medium
when produced by recombinant DNA techniques, or chemical precursors
or other chemicals when chemically synthesized. Such chondroitinase
ABC protein is also characterized as being essentially free of all
other P. vulgaris proteins. Accordingly, an isolated chondroitinase
ABC protein is produced recombinantly or synthetically and is
substantially free of cellular material and culture medium or
substantially free of chemical precursors or other chemicals and is
essentially free of all other P. vulgaris proteins.
Fragments of chondroitinase ABC which depolymerize chondroitin A,
B, or C (referred to herein as catalytically-active fragments) may
be obtained, for example, by screening peptides recombinantly
produced from the corresponding fragment of the nucleic acid
sequence of chondroitinase ABC coding for such peptides. In
addition, fragments can be chemically synthesized using techniques
known in the art such as by conventional Merrifield solid phase
f-Moc or t-Boc chemistry. For example, the chondroitinase ABC
protein may be arbitrarily divided into fragments of desired
length. The fragments can be produced (recombinantly or by chemical
synthesis) and tested to determine their enzymatic activity, for
example, by contacting the fragment with chondroitin A, B, or C
under conditions which allow for depolymerization and determining
the extent to which depolymerization occurs.
It is possible to modify the structure of the chondroitinase ABC
protein for such purposes as increasing solubility, enhancing
therapeutic efficacy, or stability (e.g., shelf life ex vivo and
resistance to proteolytic degradation in vivo). Such modified
proteins or analogues are considered functional equivalents of the
chondroitinase ABC protein as defined herein.
To facilitate purification and potentially increase solubility of
the chondroitinase ABC protein, it is possible to add an amino acid
reporter group to the protein backbone. For example, hexa-histidine
can be added to the protein for purification by immobilized metal
ion affinity chromatography (Hochuli, E. et al., (1988)
Bio/Technology 6:1321-1325). In addition, to facilitate isolation
of chondroitinase ABC protein free of irrelevant sequences,
specific endoprotease cleavage sites can be introduced between the
sequences of the reporter group and the protein or peptide.
Another aspect of the invention pertains to an antibody
specifically reactive with chondroitinase ABC. The antibodies of
this invention can be used to isolate the naturally-occurring or
native form of chondroitinase ABC or to neutralize the enzyme so
that it is unable to depolymerize chondroitin. For example, by
using isolated chondroitinase ABC protein based on the cDNA
sequence of chondroitinase ABC, anti-protein/anti-peptide antisera
or monoclonal antibodies can be made using standard methods. A
mammal such as a mouse, a hamster or a rabbit can be immunized with
an immunogenic form of the isolated chondroitinase ABC protein
(e.g., chondroitinase ABC protein or an antigenic fragment which is
capable of eliciting an antibody response). Techniques for
conferring immunogenicity on a protein or peptide include
conjugation to carriers or other techniques well known in the art.
The chondroitinase ABC protein or fragment thereof can be
administered in the presence of adjuvant. The progress of
immunization can be monitored by detection of antibody titers in
plasma or serum standard ELISA or other immunoassay can be used
with the immunogen as antigen to assess the levels of
antibodies.
Following immunization, anti-chondroitinase ABC antisera can be
obtained and, if desired, polyclonal anti-chondroitinase ABC
antibodies isolated from the serum. To produce monoclonal
antibodies, antibody producing cells (lymphocytes) can be harvested
from an immunized animal and fused by standard somatic cell fusion
procedures with immortalizing cells such as myeloma cells to yield
hybridoma cells. Such techniques are well known in the art, for
example the hybridoma technique originally developed by Kohler and
Milstein, Nature (1975) 256:495-497, as well as other techniques
such as the human B-cell hybridoma technique (Kozbar et al.,
Immunology Today (1983) 4:72) and the EBV-hybridoma technique to
produce human monoclonal antibodies (Cole et al., Monoclonal
Antibodies and Cancer Therapy (1985) Alan R. Liss, Inc. pp. 77-96).
Hybridoma cells can be screened immunochemically for production of
antibodies specifically reactive with the chondroitinase ABC
protein and the monoclonal antibodies isolated.
The term antibody as used herein is intended to include fragments
thereof which are also specifically reactive with the
chondroitinase ABC protein or fragment thereof. Antibodies can be
fragmented using conventional techniques and the fragments screened
for utility in the same manner as described above for whole
antibodies. For example, F(ab').sub.2 fragments can be generated by
treating the antibody with pepsin. The resulting F(ab').sub.2
fragment can be treated with papain to reduce disulfide bridges to
produce Fab' fragments. The antibody of the present invention is
further intended to include bispecific and chimeric molecules
having an anti-chondroitinase ABC portion.
This invention provides therapeutic compositions for the treatment
of intervertebral displacement or nerve damage. The composition
comprises a therapeutically active amount of chondroitinase ABC
protein and a pharmaceutically acceptable carrier. Administration
of the therapeutic compositions of the present invention to an
individual to be treated can be carried out using known procedures,
at dosages and for periods of time effective to depolymerize
chondroitin A, B, or C. A therapeutically active amount of
chondroitinase ABC protein may vary according to factors such as
the amount of chondroitin to be eliminated, the age, sex, and
weight of the individual, and the ability of the chondroitinase ABC
protein to depolymerize the chondroitin. Dosage regima may be
adjusted to provide the optimum therapeutic response.
The active compound (i.e., chondroitinase ABC protein) may be
administered in a convenient manner such as by injection
(subcutaneous, intravenous, etc.). If the active compound is
administered by injection, for example, about 100 units of active
compound (i.e., chondroitinase ABC protein) per dosage unit may be
administered to treat intervertebral disc displacement. One unit is
the amount of enzyme needed to mediate the release of one micromole
of 4,5 unsaturated disaccharide from a substrate of chondroitin C
sulfate per minute at 37.degree. C., pH 6.0.
The active compound may be administered parenterally. Dispersions
can be prepared in glycerol, liquid polyethylene glycols, and
mixtures thereof and in oils. Under ordinary conditions of storage
and use, these preparations may contain a preservative to prevent
the growth of microorganisms.
Pharmaceutical compositions suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersion. In all cases, the composition
must be sterile and must be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), suitable
mixtures thereof, and vegetable oils. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate or
gelatin.
Sterile injectable solutions can be prepared by incorporating
active compound (i.e., chondroitinase ABC protein) in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle which contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying which yields a
powder of the active ingredient (i.e., protein) plus any additional
desired ingredient from a previously sterile-filtered solution
thereof.
As used herein "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active compound, use thereof in
the therapeutic composition is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions
in dosage unit form for ease of administration and uniformity of
dosage. Dosage unit form as used herein refers to physically
discrete units suited as unitary dosages for the mammalian subjects
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the
elimination of chondroitin A, B, or C.
Isolated chondroitinase ABC protein (i.e., chondroitinase ABC
produced recombinantly or by chemical synthesis) is essentially
free of all other P. vulgaris proteins. Such protein is of a
consistent, well-defined composition and biological activity for
use in preparations which can be administered for therapeutic
purposes (e.g., to treat intervertebral disc displacement). Such
proteins can also be used as diagnostic reagents or in the study of
the mechanism of chondroitinase ABC and to design modified
derivatives or analogs useful in the depolymerization of
chondroitin.
This invention also provides a method of treating intervertebral
disc displacement by chemonucleolysis using isolated chondroitinase
ABC. Chondroitinase ABC is a particularly useful enzyme for the
selective chemonucleolysis of the nucleus pulposus (See, for
example, U.S. Pat. No. 4,696,816). The nucleus pulposus is made up
of proteoglycans and collagen fibers. Chondroitinase ABC attacks
the polysaccharide side chains of the proteoglycans and reduces the
swelling of the disc without affecting the structural collagen
components or degrading the protein element of the proteoglycan.
The disc then shrinks and pressure on the spinal cord is relieved.
Thus, to treat intervertebral disc displacement, an active amount
of the chondroitinase ABC protein of the invention can be applied
to the affected area. For example, 100 units of isolated
chondroitinase ABC can be injected into the center of a disc by the
standard technique of intradiscal injection (Brown, Intradiscal
Therapy, Year Book Medical Publishers, Inc., Chicago, 1983).
The invention further provides a method of treating nerve damage by
applying an active amount of the chondroitinase ABC protein of the
invention to the affected area to degrade chondroitin-6-sulfate
proteoglycans. It has been found that chondroitin 6-sulfate
proteoglycans inhibit regeneration of neurites in the adult
vertebrate central nervous system (McKeon et al., J. Neurosci
11:3398-3411 (1991)). By removing chondroitin 6-sulfate
proteoglycans from the point of injury, it is possible to promote
neurite regeneration. For example, a therapeutically effective
amount of isolated chondroitinase ABC can be applied to the point
of injury in an individual to degrade inhibitory chondroitin
6-sulfate proteoglycans. More than one dose may be administered as
indicated by the exigencies of the therapeutic situation.
The chondroitinase ABC protein of the invention can also be used as
a diagnostic reagent for detecting the presence of a
galactosaminoglycan, such as chondroitin sulfate. For example, the
chondroitinase ABC protein can be used as a reagent for determining
or quantitating the amount of galactosaminoglycan in a mammalian
tissue, such as skin, cornea, bone or cartilage (See e.g., Linker,
A. et al. (1960) J. Biol. Chem. 235: 3061-3065; Saito, H. et al.
(1968) J. Biol. Chem. 243: 1536-1542; Pettipher, E. R. et al.
(1989) Arthritis Rheum. 32: 601-607; Caterson, B. et al. (1990) J.
Cell Science 97: 411-417; and Seibel, M. J. et al. (1992) Arch.
Biochem. Biophys. 296: 410-418). To determine the presence of
chondroitin sulfate in a mammalian tissue, chondroitinase ABC
protein can be contacted with a sample of the tissue and the
presence or amount of chondroitin sulfate determined using methods
well known in the art.
The invention is further illustrated by the following examples
which should not be construed as further limiting the subject
invention. The contents of all references and published patent
applications cited throughout this application are hereby
incorporated by reference. The following methods and materials were
used throughout the examples discussed below.
Materials and Methods
Bacterial strains, plasmid and phage P. vulgaris IFO3988 was
provided by the Institute for Fermentation, Osaka, Japan. E. coli
P2392 (hsdR514(r.sup.k-, m.sup.k+), supE44, supF58, lacY1 or
(lacIZY), galT22, metB1, trpR55, (P2)) was used as the lysogen for
P2 phage. EMBL3 vector was purchased from Toyobo Co., Ltd., Japan.
PCR products were ligated with pT7 Blue T-vector(Takara Shuzo Co.,
Ltd., Japan). E. coli JM109(recA1, endA1, gyrA96, thi,
hsdR17(r.sup.k-, m.sup.k+), supE44, relA1, .lamda.-,
.DELTA.(lac-proB), (F', proAB, lacIq M15, traD36) was used as the
host strain for pMC1871 promoter selection vector (Pharmacia LKB,
Japan). E. coli XL1-Blue(endA1, hsdR17(r.sup.k-, m.sup.k+), supE44,
thi-1, recA1, gyrA96, relA1, A(lac), (F', proAB, lac,
(lacZ.DELTA.M15, Tn10(tetr)) (Int'l Dep. No. FERM BP-4170). E. coli
XL1-Blue is a host cell for both pSTV28, and pSTV29 (Takara Shuzo
Co., Ltd., Japan).
N-terminal amino acid sequence Chondroitinase ABC was purified as
described previously (Sato, N. et al. (1986) Agric. Biol. Chem. 50:
1057-1059). The N-terminal amino acid sequence of chondroitinase
ABC was sequenced by automatic Edman degradation on a gas-phase
sequencer (Applied Biosystem, Foster, Calif.). The sequence of the
N-terminal region of chondroitinase ABC was
Ala-Thr-Ser-Asn-Pro-Ala-Phe-Asp-Pro-Lys-Asn-Leu-Met-Gln-Ser-Glu-Ile-Tyr
(18 amino acid residues) (SEQ ID NO:3) The double stranded DNA
sequence is shown in FIG. 1-B (SEQ ID NOS:12-13).
Isolation of DNA and synthesis of nucleic acid, primer, and probe
Isolation of chromosomal DNA of P. vulgaris was carried out by the
standard method (Silhavy, T. J. et al. (1984) Experiments with Gene
Fusion, Cold Spring Harbor Laboratory Press). Oligonucleotides used
as primers and probe were synthesized with the DNA synthesizer,
Cyclone Plus (Milligene/Biosearch, Bedford, Mass.).
Construction and screening of the gene library SauIII AI-partially
digested fragments of total DNA were ligated to the BamHI site in
.lamda.EMBL3 arms according to Frischauf et al. (J. Mol. Biol. 170:
827-842 (1983)). The ligation mixture was packaged in vitro and
transfected to E. coli P2392 according to the instructions of the
suppliers (Stratagene, La Jolla, Calif.).
PCR amplification Primers for the chondroitinase ABC gene were
designed according to the amino acid sequence of the chondroitinase
ABC N-terminal region (SEQ ID NO:3)(FIG. 1-A). The primers were as
follows 5'-GCNACNUCNAAYCCNGC-3' (P-1, sense)(SEQ ID NO:5);
5'-GCNACNAGYAAYCCNGC-3' (P-2, sense)(SEQ ID NO:6);
5'-UACGUYAGNCUYUADAU-3' (P-3, antisense)(SEQ ID NO:7);
5'-UACGUYUCRCUYUADAU-3' (P-4 antisense)(SEQ ID NO:8) (FIG.
1-A).
PCR was performed using a GeneAmp Kit (Takara Shuzo Co., Ltd.,
Japan) in a final volume of 100 .mu.l which contained: 1 .mu.g of
genomic DNA solution, 10 .mu.l of 10.times. PCR reaction buffer, 16
.mu.l of 1.25 mM dNTP mixture, 0.6 nmol of mixed primers and 2.5
units of Taq DNA polymerase (Takara Shuzo Co., Ltd., Japan). The
mixture was subjected to PCR amplification using the DNA thermal
cycler (GeneAmp PCR System 9600, Perkin-Elmer/Cetus, Norwalk,
Conn.) for 28 cycles. Each cycle was 1 minute at 93.degree.
C.(denaturation), 1.5 minutes at 50.degree. C.(annealing) and 0.5
minute at 72.degree. C.(elongation). PCR products were analyzed by
electrophoresis through a 5% agarose gel (Nusieve GTG agarose, FMC
Bioproducts, Rockford, Me.) and the 54 bp fragment encoding 17
amino acids of N-terminal region was cut out of the gel.
Gel-purified PCR products were directly cloned into pT7 Blue PCR
vector.
DNA Sequencing and Isolation of the Chondroitinase ABC Gene
Double-stranded plasmids purified by polyethylene glycol were
denatured with alkali and sequenced by dideoxynucleotide chain
termination method (Sanger, R. et al. (1977) Proc. Natl. Acad. Sci.
USA 74: 5463-5467) using the sequence system, Hitachi WS10A
Personal Sequencer (Hitachi Electronics Co., Ltd., Japan) Direct
sequencing was done according to the method of Gyllesten &
Erlich (Gyllensten, U. (1989) in PCR Technology, Erlich, H. A.,
Ed., Stockton Press, New York, pp. 45-60). PCR screening was
carried out by the method of Olson et al.(Science 245: 1434-1435
(1989)). Plaque hybridization (Sambrook, J. et al. (1989) Molecular
Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor
Laboratory Press) and Southern hybridization (Southern, E. M.
(1975) J. Mol. Biol. 8: 503-517) were performed as outlined in the
instructions of the supplier(Amersham Japan).
Primer extension analysis A 21-mer oligonucleotide(5'-CTA ATG GGT
TAT TTT GTG CAA-3') (SEQ ID NO:4) complementary to the 5'-end
(nucleotides 355-375) of the chondroitinase ABC gene was used as a
primer. It was labeled with .gamma.-.sup.32P ATP (Amersham Japan)
using polynucleotide kinase (Toyobo Co., Ltd., Japan). Total RNA of
P. vulgaris was prepared according to the method of Aiba (J. Biol.
Chem. 260: 3063-3070 (1985)). The labeled primer and 5 .mu.g of
total RNA were coprecipitated with ethanol. After annealing at
25.degree. C. for 6 hours in a hybridization buffer (80% formamide,
40 mM PIPES(pH 6.4), 1 mM EDTA and 400 mM NaCl), 250 mM NaCl, 50 mM
sodium acetate(pH 4.6), 4.5 mM ZnSO.sub.4, 100 .mu.g/ml
heat-denatured salmon testes DNA and 15 unit/.mu.l reverse
transcriptase of Rous associated virus 2 (Takara Shuzo Co., Ltd.,
Japan) were added to the mixture. The primer extension reaction was
carried out at 37.degree. C. for 60 minutes.
Culture conditions Cells of E. coli XL1-Blue carrying recombinant
plasmid were grown in 3 ml of LB broth(1% tryptone, 0.5% yeast
extract, 1% NaCl, 25 .mu.g/ml of chloramphenicol (pH 7.5)) at
37.degree. C. for 16 hr with reciprocation (120 rpm, 5 cm stroke).
The cells were harvested by centrifugation and washed twice with
0.85% saline solution. Cells were transferred to 100 ml of
chondroitin 6-sulfate (Taiyo Fishery Co., Ltd., Japan) medium(0.7%
K.sub.2HPO.sub.4, 0.3% KH.sub.2PO.sub.4, 0.01%
MgSO.sub.4.7H.sub.2O, 0.1% (NH.sub.4).sub.2SO.sub.4, 0.1% yeast
extract, 0.3% chondroitin 6-sulfate, 0.01% glucose, 25 .mu.g/ml
chloramphenicol (pH 7.5)) or glucose medium (composition is the
same as that of chondroitin medium except that glucose (0.3%) was
used as a carbon source) to make a final concentration of
A.sub.610=0.05. After incubation for 3 days at 37.degree. C. with
reciprocation, the cells were removed by centrifugation and
degradation products of chondroitin 6-sulfate in the culture fluid
were determined. The cells harvested from chondroitin and glucose
media were washed twice with 50 mM Tris-HCl buffer (pH 8.0) and
sonicated at 90 kHz for 5 minutes at 0.degree. C. The cell debris
were removed by centrifugation at 20,000 g for 30 minutes, and the
supernatant was used for the assay of chondroitinase ABC.
Enzyme assay Chondroitinase ABC was assayed as described previously
(Sato, N. et al. (1986) J. Ferment. Technol. 64: 155-159). The
assay mixture (3 ml) containing 0.5% chondroitin 6-sulfate, 100 mM
potassium phosphate buffer (pH 8.0) and cell extract, was incubated
at 37.degree. C. for 10 minutes, and the amount of
N-acetylgalactosamine end group formed was determined by the method
of Reissig (J. Biol. Chem. 217: 959-966 (1955)). Activity was
expressed as the quantity of enzyme that catalyzed the formation of
1 .mu.mol of unsaturated disaccharide (.DELTA.di-6S) from
chondroitin 6-sulfate per minute at 37.degree. C.
Western blot analysis IgG specific to chondroitinase ABC was
isolated from antisera raised in guinea pig using the technique
described previously (Sato, N. et al. (1988) Biotechnol. Appl.
Biochem. 10: 385-393). Proteins in crude cell extracts prepared
from E. coli transformant were separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described
previously (Sato, N. et al. (1986) Agric. Biol. Chem. 50:
1057-1059). Western blotting procedures were described previously
(Sato, N. et al. (1989) Appl. Microbiol. Biotechnol. 30:
153-159).
EXAMPLE 1
Isolation and Sequence Determination of the Chondroitinase ABC
Gene
According to the amino acid sequence of the N-terminal region of
purified chondroitinase ABC
(Ala-Thr-Ser-Asn-Pro-Ala-Phe-Asp-Pro-Lys-Asn-Leu-Met-Gln-Ser-Glu-Ile-Tyr
(FIG. 1-A)(SEQ ID NO:3)), a set of degenerate oligo mixed primers
(5'-GCNACNUCNAAYCCNGC-3' (P-1, sense)(SEQ ID NO:5);
5'-GCNACNAGYAAYCCNGC-3' (P-2, sense)(SEQ ID NO:6);
5'-UACGUYAGNCUYUADAU-3' (P-3, antisense)(SEQ ID NO:7);
5'-UACGUYUCRCUYUADAU-3' (P-4 antisense)(SEQ ID NO:8))(FIG. 1-A)
were synthesized as follows. To determine the appropriate primers
for sequencing, PCR amplification of a combination of primers
P-1(SEQ ID NO:5), P-2(SEQ ID NO:6) (sense) and P-3(SEQ ID NO:7),
P-4(SEQ ID NO:8) (antisense) was performed. After agarose gel
electrophoresis of these PCR products, a 54 bp fragment was
extracted and directly inserted into pT7 Blue PCR vector, and the
inserted fragment was sequenced. The nucleotide sequence of this
fragment was found to be identical to the N-terminal amino acid
sequence (FIG. 1-B) (SEQ ID NO:3). Then, using primer A
(5'-GCAACCAGCAATCCTGCA-3')(SEQ ID NO:10), primer B (.[.5.].
.Iadd.3.Iaddend.'-GACTACGTCAGGCTTT.[.T.].
.Iadd.A.Iaddend.AAT-.[.3.]. .Iadd.5.Iaddend.')(SEQ ID NO:11) (FIG.
1-B) and 1 .mu.g of P. vulgaris genomic DNA as a template, PCR
analysis was performed and PCR products were analyzed by agarose
gel electrophoresis. No non-specific PCR products were
observed.
We then diluted .gamma.EMBL3 recombinant phage stock library. The
diluted library was used for PCR screening. An unique 54 bp
fragment was clearly detected until the dilution of
1/10.sup.3(2.times.10.sup.5 pfu) phage stock solution as a
template. The diluted phage solution was divided by
1/10(2.times.10.sup.4 pfu) and was infected into E. coli P2392.
They were then subjected to plaque hybridization using
.sup.32P-labeled probe (5'-CATTTGATCCTAAAAATCTGATGCA-3')(SEQ ID
NO:9)(FIG. 1-B). The recombinant phages were chosen at random and
analyzed by restriction mapping and Southern blotting. All phages
contained common 4.2 kb EcoRV-EcoRI, 1.1 kb ClaI, and 2.0 kb
EcoRV-HindIII fragments which hybridized strongly with the
probe(SEQ ID NO:9). The restriction maps of three types of SalI
fragments are shown in FIG. 1-C. Southern hybridization patterns of
restricted genomic DNA from P. vulgaris matched the restriction map
of these fragments. This result suggests that the 4.2 kb
EcoRV-EcoRI fragment originated in the P. vulgaris genome, and
therefore, the chondroitinase ABC gene exists as a single copy.
When purified chondroitinase ABC from P. vulgaris was analyzed by
SDS-PAGE, two types of chondroitinase ABC protein, one 100 kd
protein and one subunit-like protein at 80 kd and 20 kd, were
observed. The amino acid composition of the 100 kd protein and the
subunit-like protein (80 kd and 20 kd) were quite similar, and the
N-terminal amino acid sequences of the 100 kd and 20 kd proteins
were identical. The results indicate that the two forms of
chondroitinase ABC were not derived from two separate
chondroitinase ABC genes.
The 5.2 kb SalI-EcoRI fragment in the recombinant .gamma.EMBL3 (No.
11-5) (FIG. 1-C) was subcloned into pSTV29 for sequencing and the
resulting hybrid plasmid was designated pCHS6. The entire 3,063 bp
nucleotide sequence of the coding region for the chondroitinase ABC
gene as well as 224 and 200 nucleotides of the upstream and
downstream regions, respectively, and the deduced amino acid
sequence of chondroitinase ABC are shown in FIG. 7 (SEQ ID NO:1).
The 25-mer oligonucleotide probe (SEQ ID NO:9) hybridized to
nucleotide 314-337. The 16/18 nucleotide of primer A and the 17/18
nucleotide of primer B were the same in nucleotides 297-313 and
333-349. The G+C content of the chondroitinase ABC gene was 38.6%.
The open reading frame encoded a polypeptide with a molecular
weight of 115,218, which represents a precursor polypeptide
containing a signal peptide sequence that is subsequently cleaved
off at Ala.sup.24-Ala.sup.25 during secretion of the mature
chondroitinase ABC protein having a molecular weight of
112,365.
EXAMPLE 2
Analysis of the transcription region of the chondroitinase ABC gene
In order to confirm the potential promoter region of the
chondroitinase ABC gene, we amplified the region of nucleotide
112-283 using PCR. The PCR product was blunt-ended with T4 DNA
polymerase and inserted into the SmaI site of the promoter
selection vector, pMC 1871, and the hybrid plasmid, designated
pCHSP, was introduced into E. coli JM109 (FIG. 2)(SEQ ID NO:14).
The transformant was then cultured in an LB medium containing 25
.mu.g/ml tetracycline at 37.degree. C. for 14 hr, and
.beta.-galactosidase activity was assayed (Table I). Although the
.beta.-galactosidase activity of the E. coli transformant carrying
pMC1871 was not detectable, the E. coli transformant carrying pCHSP
produced .beta.-galactosidase. This result indicates that the
chondroitinase ABC gene can function as a promoter in E. coli
cells. However, there is a possibility that the promoter recognized
in E. coli cells may not be the promoter in P. vulgaris. To confirm
that the promoter is recognized in P. vulgaris, primer extension
analysis was carried out (FIG. 3) (SEQ ID NO:15). The transcription
start point was localized to an adenosine 41 bp upstream from the
start codon, ATG (FIG. 4) (SEQ ID NO:16). The potential pribnow box
(TTTAAT) (nucleotides 169-174) was located 12 bp upstream from the
transcription start point (FIG. 4) (SEQ ID NO: 16). However, the
-35 consensus sequence was not found near 35 bp upstream of the
start point except for 47 bp upstream of the start point (TAGGCA)
(FIG. 4) (SEQ ID NO:16). The Shine-Dalgarno ribosomal binding site
(AGGAGA) (nucleotides 213-218) was found 9 bp upstream from the
initiation codon, ATG (FIG. 4) (SEQ ID NO: 16). A terminator-like
palindrome sequence consisting of an 11 nucleotide stem with a 4
nucleotide loop structure (stacking energy 24 kcal/mol) was located
9 nucleotides downstream from the stop codon, TGA (FIG. 4) (SEQ ID
NO:16). Judging from the secondary structure prediction, this
stem-loop structure resembles a .sigma.-dependent transcription
terminator.
TABLE-US-00001 TABLE I .beta.-Galactosidase productivity of E-coli
transformants .beta.-Galactosidase activity Strain Activity
Specific activity/ E. coli JM109 (U/mi-culture) (U/mg-protein)
/pMC1871 0 0 /pCHSP 0.2 0.4 1 U is defined as the amount that
produced 1 .mu.mol of .alpha.-nitrophenol per h.
EXAMPLE 3
Production of chondroitinase ABC from E. coli transformant To
demonstrate that the isolated gene codes for chondroitinase ABC, we
constructed pCHS.DELTA.6 and pCHS26 (FIG. 5). pCHS.DELTA.6 was
constructed by removing the SalI-EcoRV region (about 1 kb) upstream
from the promoter region from the chondroitinase ABC gene. While
pCHS26 was constructed by removing the HindIII-EcoRI region which
corresponded to about one third of the 3'-terminal region of the
chondroitinase ABC structural gene. These plasmids (pCHS6,
pCHS.DELTA.6 and pCHS26) were introduced into E. coli XL 1-Blue,
and E. coli transformants were cultured in chondroitin or glucose
medium, and chondroitinase ABC activities were assayed using the
crude extract. The culture fluids of the chondroitin medium were
also analyzed to determine degradation products of chondroitin
6-sulfate (Table II). The E. coli transformant carrying pCHS6
(containing a 1.0 kb fragment upstream from the promoter) produced
the chondroitinase ABC when cultured in chondroitin medium,
however, no chondroitinase ABC activity was observed when the
transformant was cultured in glucose medium. In contrast, the E.
coli transformant carrying pCHS.DELTA.6 produced chondroitinase ABC
when cultured in either chondroitin or glucose media. The
production levels of chondroitinase ABC, cultured in chondroitin
media, were 2.6 fold(/pCHS6) and 187 fold(/pCHS.DELTA.6) higher
than that of P. vulgaris. Even cultured in glucose medium, the
production level of chondroitinase ABC in the E. coli transformant
carrying pCHS.DELTA.6 was 187 fold higher than that of P. vulgaris
cultured in chondroitin medium. This result suggests that the
regulatory sequence might be in the SalI-EcoRV region. Although
chondroitin 6-sulfate added to the medium was degraded (p/CHS6 and
/pCHS.DELTA.6), E. coli transformants were not able to utilize
chondroitin sulfate as a carbon source.
TABLE-US-00002 TABLE II Chondroitinase ABC Activity of E. coli
Transformants Intracellular chondroitinase ABC activity Chondroitin
medium Glucose Cultured medium (0.3%) medium (0.3%) Amount of
4,5.DELTA. Specific.sup.b Specific chondroitin-6 Strain
Activity.sup.a activity Actiity activity (.mu.g/ml-culture) E. coli
0 0 0 0 0 XL1-Blue /pSTV29 0 0 0 0 0 /pCHS6 4.1 .times. 10.sup.-3
1.6 .times. 10.sup.-2 0 0 192.7 /pCHS26 0 0 0 0 0 /pCHS.DELTA.6 0.3
1.2 0.3 0.5 1542.4 P. vulgaris 1.6 .times. 10.sup.-3 1.2 .times.
10.sup.-2 0 0 1738.4 .sup.a1 U: enzyme activity producing 1
.mu.mol, 4,5.DELTA. chondroitin-6 per min .sup.bU/mg-protein
It has been reported that the Bacteriodes thetaiotaomicron
chondroitin lyase II gene is adjacent to the chondrosulfatase gene
which may be a part of an operon (Guthrie, E. P. et al. (1987) J.
Bacteriol. 169: 1192-1199). These same investigators reported that
the promoter for this gene recognized in E. coli may not be the
promoter from which the chondroitin lyase II gene is transcribed
from in B. thetaiotaomicron (Ld.) In fact, a putative open reading
frame 12 bp upstream from the initiation codon, ATG, was found in
the chondroitinase ABC gene (FIG. 4) (SEQ ID NO: 16). However,
primer extension analysis revealed that the transcription start
point is located 41 bp upstream from the initiation codon in P.
vulgaris(FIG. 3) (SEQ ID NO: 15). Even though the chondroitinase
ABC gene from P. vulgaris cells was also part of an operon,
chondroitinase ABC gene was transcribed 41 bp upstream from the
initiation codon in P. vulgaris cells.
The secondary structure of chondroitinase ABC was estimated by the
method of Chou and Fasman (Annu. Rev. Biochem. 47: 251-276 (1978)).
A highly complex region was found between amino acid residues 450
and 850. The pCHS26 lacks one-third of the chondroitinase ABC gene
encoding the C-terminal region (amino acid residues 646-1021).
Removing this region of the enzyme caused the disappearance of
chondroitinase ABC activity (Table II). This result suggests that
there might be an active site in this region.
Recombinant chondroitinase ABC produced by E. coli carrying
pCHS.DELTA.6 was analyzed by SDS-PAGE followed by immunoblotting
(FIG. 6). The immunoblotting patterns of recombinant and native
chondroitinase ABC (100 kd) were quite similar. Our previous report
showed chondroitinase ABC purified from P. vulgaris was a subunit
structure consisting of a 90 kd and a 20 kd protein by SDS-PAGE
(Sato, N. et al. (1986) Agric. Biol. Chem. 50: 1057-1059), because
this subunit protein would not be separated even using gel
filtration and other chromatographic techniques. However, by
analysis of the N-terminal sequence, we found that the 100 kd
protein and the 20 kd protein had the same N-terminal amino acid
sequence. By immunoblot analysis, the 80 kd protein also reacts
with IgG specific to the 100 kd protein. Furthermore, genomic
restriction analysis suggested that chondroitinase ABC gene was a
single gene. When we extracted the 100 kd band of chondroitinase
ABC from the acrylamide gel and electrophoresed it again in
SDS-PAGE, 80 kd and 20 kd bands appeared. The purified
chondroitinase ABC contained no protease activity. These results
suggest that chondroitinase ABC was partially digested not
enzymatically, but physically in the course of sample preparation
for SDS-PAGE.
Equivalents
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, numerous equivalents to
the specific procedures described herein. Such equivalents are
considered to be within the scope of this invention and are covered
by the following claims.
SEQUENCE LISTINGS
1
1713066DNAProteus vulgarisCDS(1)..(3063) 1atg ccg ata ttt cgt ttt
act gca ctt gca atg aca ttg ggg cta tta 48Met Pro Ile Phe Arg Phe
Thr Ala Leu Ala Met Thr Leu Gly Leu Leu1 5 10 15tca gcg cct tat aac
gcg atg gca gcc acc agc aat cct gca ttt gat 96Ser Ala Pro Tyr Asn
Ala Met Ala Ala Thr Ser Asn Pro Ala Phe Asp 20 25 30cct aaa aat ctg
atg cag tca gaa att tac cat ttt gca caa aat aac 144Pro Lys Asn Leu
Met Gln Ser Glu Ile Tyr His Phe Ala Gln Asn Asn 35 40 45cca tta gca
gac ttc tca tca gat aaa aac tca ata cta acg tta tct 192Pro Leu Ala
Asp Phe Ser Ser Asp Lys Asn Ser Ile Leu Thr Leu Ser 50 55 60gat aaa
cgt agc att atg gga aac caa tct ctt tta tgg aaa tgg aaa 240Asp Lys
Arg Ser Ile Met Gly Asn Gln Ser Leu Leu Trp Lys Trp Lys65 70 75
80ggt ggt agt agc ttt act tta cat aaa aaa ctg att gtc ccc acc gat
288Gly Gly Ser Ser Phe Thr Leu His Lys Lys Leu Ile Val Pro Thr Asp
85 90 95aaa gaa gca tct aaa gca tgg gga cgc tca tct acc ccc gtt ttc
tca 336Lys Glu Ala Ser Lys Ala Trp Gly Arg Ser Ser Thr Pro Val Phe
Ser 100 105 110ttt tgg ctt tac aat gaa aaa ccg att gat ggt tat ctt
act atc gat 384Phe Trp Leu Tyr Asn Glu Lys Pro Ile Asp Gly Tyr Leu
Thr Ile Asp 115 120 125ttc gga gaa aaa ctc att tca acc agt gag gct
cag gca ggc ttt aaa 432Phe Gly Glu Lys Leu Ile Ser Thr Ser Glu Ala
Gln Ala Gly Phe Lys 130 135 140gta aaa tta gat ttc act ggc tgg cgt
gct gtg gga gtc tct tta aat 480Val Lys Leu Asp Phe Thr Gly Trp Arg
Ala Val Gly Val Ser Leu Asn145 150 155 160aac gat ctt gaa aat cga
gag atg acc tta aat gca acc aat acc tcc 528Asn Asp Leu Glu Asn Arg
Glu Met Thr Leu Asn Ala Thr Asn Thr Ser 165 170 175tct gat ggt act
caa gac agc att ggg cgt tct tta ggt gct aaa gtc 576Ser Asp Gly Thr
Gln Asp Ser Ile Gly Arg Ser Leu Gly Ala Lys Val 180 185 190gat agt
att cgt ttt aaa gcg cct tct aat gtg agt cag ggt gaa atc 624Asp Ser
Ile Arg Phe Lys Ala Pro Ser Asn Val Ser Gln Gly Glu Ile 195 200
205tat atc gac cgt att atg ttt tct gtc gat gat gct cgc tac caa tgg
672Tyr Ile Asp Arg Ile Met Phe Ser Val Asp Asp Ala Arg Tyr Gln Trp
210 215 220tct gat tat caa gta aaa act cgc tta tca gaa cct gaa att
caa ttt 720Ser Asp Tyr Gln Val Lys Thr Arg Leu Ser Glu Pro Glu Ile
Gln Phe225 230 235 240cac aac gta aag cca caa cta cct gta aca cct
gaa aat tta gcg gcc 768His Asn Val Lys Pro Gln Leu Pro Val Thr Pro
Glu Asn Leu Ala Ala 245 250 255att gat ctt att cgc caa cgt cta att
aat gaa ttt gtc gga ggt gaa 816Ile Asp Leu Ile Arg Gln Arg Leu Ile
Asn Glu Phe Val Gly Gly Glu 260 265 270aaa gag aca aac ctc gca tta
gaa gag aat atc agc aaa tta aaa agt 864Lys Glu Thr Asn Leu Ala Leu
Glu Glu Asn Ile Ser Lys Leu Lys Ser 275 280 285gat ttc gat gct ctt
aat att cac act tta gca aat ggt gga acg caa 912Asp Phe Asp Ala Leu
Asn Ile His Thr Leu Ala Asn Gly Gly Thr Gln 290 295 300ggc aga cat
ctg atc act gat aaa caa atc att att tat caa cca gag 960Gly Arg His
Leu Ile Thr Asp Lys Gln Ile Ile Ile Tyr Gln Pro Glu305 310 315
320aat ctt aac tcc caa gat aaa caa cta ttt gat aat tat gtt att tta
1008Asn Leu Asn Ser Gln Asp Lys Gln Leu Phe Asp Asn Tyr Val Ile Leu
325 330 335ggt aat tac acg aca tta atg ttt aat att agc cgt gct tat
gtg ctg 1056Gly Asn Tyr Thr Thr Leu Met Phe Asn Ile Ser Arg Ala Tyr
Val Leu 340 345 350gaa aaa gat ccc aca caa aag gcg caa cta aag cag
atg tac tta tta 1104Glu Lys Asp Pro Thr Gln Lys Ala Gln Leu Lys Gln
Met Tyr Leu Leu 355 360 365atg aca aag cat tta tta gat caa ggc ttt
gtt aaa ggg agt gct tta 1152Met Thr Lys His Leu Leu Asp Gln Gly Phe
Val Lys Gly Ser Ala Leu 370 375 380gtg aca acc cat cac tgg gga tac
agt tct cgt tgg tgg tat att tcc 1200Val Thr Thr His His Trp Gly Tyr
Ser Ser Arg Trp Trp Tyr Ile Ser385 390 395 400acg tta tta atg tct
gat gca cta aaa gaa gcg aac cta caa act caa 1248Thr Leu Leu Met Ser
Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln 405 410 415gtt tat gat
tca tta ctg tgg tat tca cgt gag ttt aaa agt agt ttt 1296Val Tyr Asp
Ser Leu Leu Trp Tyr Ser Arg Glu Phe Lys Ser Ser Phe 420 425 430gat
atg aaa gta agt gct gat agc tct gat cta gat tat ttc aat acc 1344Asp
Met Lys Val Ser Ala Asp Ser Ser Asp Leu Asp Tyr Phe Asn Thr 435 440
445tta tct cgc caa cat tta gcc tta tta tta cta gag cct gat gat caa
1392Leu Ser Arg Gln His Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln
450 455 460aag cgt atc aac tta gtt aat act ttc agc cat tat atc act
ggc gca 1440Lys Arg Ile Asn Leu Val Asn Thr Phe Ser His Tyr Ile Thr
Gly Ala465 470 475 480tta acg caa gtg cca ccg ggt ggt aaa gat ggt
tta cgc cct gat ggt 1488Leu Thr Gln Val Pro Pro Gly Gly Lys Asp Gly
Leu Arg Pro Asp Gly 485 490 495aca gca tgg cga cat gaa ggc aac tat
ccg ggc tac tct ttc cca gcc 1536Thr Ala Trp Arg His Glu Gly Asn Tyr
Pro Gly Tyr Ser Phe Pro Ala 500 505 510ttt aaa aat gcc tct cag ctt
att tat tta tta cgc gat aca cca ttt 1584Phe Lys Asn Ala Ser Gln Leu
Ile Tyr Leu Leu Arg Asp Thr Pro Phe 515 520 525tca gtg ggt gaa agt
ggt tgg aat aac ctg aaa aaa gcg atg gtt tca 1632Ser Val Gly Glu Ser
Gly Trp Asn Asn Leu Lys Lys Ala Met Val Ser 530 535 540gcg tgg atc
tac agt aat cca gaa gtt gga tta ccg ctt gca gga aga 1680Ala Trp Ile
Tyr Ser Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg545 550 555
560cac cct ttt aac tca cct tcg tta aaa tca gtc gct caa ggc tat tac
1728His Pro Phe Asn Ser Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr Tyr
565 570 575tgg ctt gcc atg tct gca aaa tca tcg cct gat aaa aca ctt
gca tct 1776Trp Leu Ala Met Ser Ala Lys Ser Ser Pro Asp Lys Thr Leu
Ala Ser 580 585 590att tat ctt gcg att agt gat aaa aca caa aat gaa
tca act gct att 1824Ile Tyr Leu Ala Ile Ser Asp Lys Thr Gln Asn Glu
Ser Thr Ala Ile 595 600 605ttt gga gaa act att aca cca gcg tct tta
cct caa ggt ttc tat gcc 1872Phe Gly Glu Thr Ile Thr Pro Ala Ser Leu
Pro Gln Gly Phe Tyr Ala 610 615 620ttt aat ggc ggt gct ttt ggt att
cat cgt tgg caa gat aaa atg gtg 1920Phe Asn Gly Gly Ala Phe Gly Ile
His Arg Trp Gln Asp Lys Met Val625 630 635 640aca ctg aaa gct tat
aac acc aat gtt tgg tca tct gaa att tat aac 1968Thr Leu Lys Ala Tyr
Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr Asn 645 650 655aaa gat aac
cgt tat ggc cgt tac caa agt cat ggt gtc gct caa ata 2016Lys Asp Asn
Arg Tyr Gly Arg Tyr Gln Ser His Gly Val Ala Gln Ile 660 665 670gtg
agt aat ggc tcg cag ctt tca cag ggc tat cag caa gaa ggt tgg 2064Val
Ser Asn Gly Ser Gln Leu Ser Gln Gly Tyr Gln Gln Glu Gly Trp 675 680
685gat tgg aat aga atg caa ggg gca acc act att cac ctt cct ctt aaa
2112Asp Trp Asn Arg Met Gln Gly Ala Thr Thr Ile His Leu Pro Leu Lys
690 695 700gac tta gac agt cct aaa cct cat acc tta atg caa cgt gga
gag cgt 2160Asp Leu Asp Ser Pro Lys Pro His Thr Leu Met Gln Arg Gly
Glu Arg705 710 715 720gga ttt agc gga aca tca tcc ctt gaa ggt caa
tat ggc atg atg gca 2208Gly Phe Ser Gly Thr Ser Ser Leu Glu Gly Gln
Tyr Gly Met Met Ala 725 730 735ttc gat ctt att tat ccc gcc aat ctt
gag cgt ttt gat cct aat ttc 2256Phe Asp Leu Ile Tyr Pro Ala Asn Leu
Glu Arg Phe Asp Pro Asn Phe 740 745 750act gcg aaa aag agt gta tta
gcc gct gat aat cac tta att ttt att 2304Thr Ala Lys Lys Ser Val Leu
Ala Ala Asp Asn His Leu Ile Phe Ile 755 760 765ggt agc aat ata aat
agt agt gat aaa aat aaa aat gtt gaa acg acc 2352Gly Ser Asn Ile Asn
Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr 770 775 780tta ttc caa
cat gcc att act cca aca tta aat acc ctt tgg att aat 2400Leu Phe Gln
His Ala Ile Thr Pro Thr Leu Asn Thr Leu Trp Ile Asn785 790 795
800gga caa aag ata gaa aac atg cct tat caa aca aca ctt caa caa ggt
2448Gly Gln Lys Ile Glu Asn Met Pro Tyr Gln Thr Thr Leu Gln Gln Gly
805 810 815gat tgg tta att gat agc aat ggc aat ggt tac tta att act
caa gca 2496Asp Trp Leu Ile Asp Ser Asn Gly Asn Gly Tyr Leu Ile Thr
Gln Ala 820 825 830gaa aaa gta aat gta agt cgc caa cat cag gtt tca
gcg gaa aat aaa 2544Glu Lys Val Asn Val Ser Arg Gln His Gln Val Ser
Ala Glu Asn Lys 835 840 845aat cgc caa ccg aca gaa gga aac ttt agc
tcg gca tgg atc gat cac 2592Asn Arg Gln Pro Thr Glu Gly Asn Phe Ser
Ser Ala Trp Ile Asp His 850 855 860agc act cgc ccc aaa gat gcc agt
tat gag tat atg gtc ttt tta gat 2640Ser Thr Arg Pro Lys Asp Ala Ser
Tyr Glu Tyr Met Val Phe Leu Asp865 870 875 880gcg aca cct gaa aaa
atg gga gag atg gca caa aaa ttc cgt gaa aat 2688Ala Thr Pro Glu Lys
Met Gly Glu Met Ala Gln Lys Phe Arg Glu Asn 885 890 895aat ggg tta
tat cag gtt ctt cgt aag gat aaa gac gtt cat att att 2736Asn Gly Leu
Tyr Gln Val Leu Arg Lys Asp Lys Asp Val His Ile Ile 900 905 910ctc
gat aaa ctc agc aat gta acg gga tat gcc ttt tat cag cca gca 2784Leu
Asp Lys Leu Ser Asn Val Thr Gly Tyr Ala Phe Tyr Gln Pro Ala 915 920
925tca att gaa gac aaa tgg atc aaa aag gtt aat aaa cct gca att gtg
2832Ser Ile Glu Asp Lys Trp Ile Lys Lys Val Asn Lys Pro Ala Ile Val
930 935 940atg act cat cga caa aaa gac act ctt att gtc agt gca gtt
aca cct 2880Met Thr His Arg Gln Lys Asp Thr Leu Ile Val Ser Ala Val
Thr Pro945 950 955 960gat tta aat atg act cgc caa aaa gca gca act
cct gtc acc atc aat 2928Asp Leu Asn Met Thr Arg Gln Lys Ala Ala Thr
Pro Val Thr Ile Asn 965 970 975gtc acg att aat ggc aaa tgg caa tct
gct gat aaa aat agt gaa gtg 2976Val Thr Ile Asn Gly Lys Trp Gln Ser
Ala Asp Lys Asn Ser Glu Val 980 985 990aaa tat cag gtt tct ggt gat
aac act gaa ctg acg ttt acg agt tac 3024Lys Tyr Gln Val Ser Gly Asp
Asn Thr Glu Leu Thr Phe Thr Ser Tyr 995 1000 1005ttt ggt att cca
caa gaa atc aaa ctc tcg cca ctc cct tga 3066Phe Gly Ile Pro Gln Glu
Ile Lys Leu Ser Pro Leu Pro 1010 1015 102021021PRTProteus vulgaris
2Met Pro Ile Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu Leu1 5
10 15Ser Ala Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe
Asp 20 25 30Pro Lys Asn Leu Met Gln Ser Glu Ile Tyr His Phe Ala Gln
Asn Asn 35 40 45Pro Leu Ala Asp Phe Ser Ser Asp Lys Asn Ser Ile Leu
Thr Leu Ser 50 55 60Asp Lys Arg Ser Ile Met Gly Asn Gln Ser Leu Leu
Trp Lys Trp Lys65 70 75 80Gly Gly Ser Ser Phe Thr Leu His Lys Lys
Leu Ile Val Pro Thr Asp 85 90 95Lys Glu Ala Ser Lys Ala Trp Gly Arg
Ser Ser Thr Pro Val Phe Ser 100 105 110Phe Trp Leu Tyr Asn Glu Lys
Pro Ile Asp Gly Tyr Leu Thr Ile Asp 115 120 125Phe Gly Glu Lys Leu
Ile Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys 130 135 140Val Lys Leu
Asp Phe Thr Gly Trp Arg Ala Val Gly Val Ser Leu Asn145 150 155
160Asn Asp Leu Glu Asn Arg Glu Met Thr Leu Asn Ala Thr Asn Thr Ser
165 170 175Ser Asp Gly Thr Gln Asp Ser Ile Gly Arg Ser Leu Gly Ala
Lys Val 180 185 190Asp Ser Ile Arg Phe Lys Ala Pro Ser Asn Val Ser
Gln Gly Glu Ile 195 200 205Tyr Ile Asp Arg Ile Met Phe Ser Val Asp
Asp Ala Arg Tyr Gln Trp 210 215 220Ser Asp Tyr Gln Val Lys Thr Arg
Leu Ser Glu Pro Glu Ile Gln Phe225 230 235 240His Asn Val Lys Pro
Gln Leu Pro Val Thr Pro Glu Asn Leu Ala Ala 245 250 255Ile Asp Leu
Ile Arg Gln Arg Leu Ile Asn Glu Phe Val Gly Gly Glu 260 265 270Lys
Glu Thr Asn Leu Ala Leu Glu Glu Asn Ile Ser Lys Leu Lys Ser 275 280
285Asp Phe Asp Ala Leu Asn Ile His Thr Leu Ala Asn Gly Gly Thr Gln
290 295 300Gly Arg His Leu Ile Thr Asp Lys Gln Ile Ile Ile Tyr Gln
Pro Glu305 310 315 320Asn Leu Asn Ser Gln Asp Lys Gln Leu Phe Asp
Asn Tyr Val Ile Leu 325 330 335Gly Asn Tyr Thr Thr Leu Met Phe Asn
Ile Ser Arg Ala Tyr Val Leu 340 345 350Glu Lys Asp Pro Thr Gln Lys
Ala Gln Leu Lys Gln Met Tyr Leu Leu 355 360 365Met Thr Lys His Leu
Leu Asp Gln Gly Phe Val Lys Gly Ser Ala Leu 370 375 380Val Thr Thr
His His Trp Gly Tyr Ser Ser Arg Trp Trp Tyr Ile Ser385 390 395
400Thr Leu Leu Met Ser Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln
405 410 415Val Tyr Asp Ser Leu Leu Trp Tyr Ser Arg Glu Phe Lys Ser
Ser Phe 420 425 430Asp Met Lys Val Ser Ala Asp Ser Ser Asp Leu Asp
Tyr Phe Asn Thr 435 440 445Leu Ser Arg Gln His Leu Ala Leu Leu Leu
Leu Glu Pro Asp Asp Gln 450 455 460Lys Arg Ile Asn Leu Val Asn Thr
Phe Ser His Tyr Ile Thr Gly Ala465 470 475 480Leu Thr Gln Val Pro
Pro Gly Gly Lys Asp Gly Leu Arg Pro Asp Gly 485 490 495Thr Ala Trp
Arg His Glu Gly Asn Tyr Pro Gly Tyr Ser Phe Pro Ala 500 505 510Phe
Lys Asn Ala Ser Gln Leu Ile Tyr Leu Leu Arg Asp Thr Pro Phe 515 520
525Ser Val Gly Glu Ser Gly Trp Asn Asn Leu Lys Lys Ala Met Val Ser
530 535 540Ala Trp Ile Tyr Ser Asn Pro Glu Val Gly Leu Pro Leu Ala
Gly Arg545 550 555 560His Pro Phe Asn Ser Pro Ser Leu Lys Ser Val
Ala Gln Gly Tyr Tyr 565 570 575Trp Leu Ala Met Ser Ala Lys Ser Ser
Pro Asp Lys Thr Leu Ala Ser 580 585 590Ile Tyr Leu Ala Ile Ser Asp
Lys Thr Gln Asn Glu Ser Thr Ala Ile 595 600 605Phe Gly Glu Thr Ile
Thr Pro Ala Ser Leu Pro Gln Gly Phe Tyr Ala 610 615 620Phe Asn Gly
Gly Ala Phe Gly Ile His Arg Trp Gln Asp Lys Met Val625 630 635
640Thr Leu Lys Ala Tyr Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr Asn
645 650 655Lys Asp Asn Arg Tyr Gly Arg Tyr Gln Ser His Gly Val Ala
Gln Ile 660 665 670Val Ser Asn Gly Ser Gln Leu Ser Gln Gly Tyr Gln
Gln Glu Gly Trp 675 680 685Asp Trp Asn Arg Met Gln Gly Ala Thr Thr
Ile His Leu Pro Leu Lys 690 695 700Asp Leu Asp Ser Pro Lys Pro His
Thr Leu Met Gln Arg Gly Glu Arg705 710 715 720Gly Phe Ser Gly Thr
Ser Ser Leu Glu Gly Gln Tyr Gly Met Met Ala 725 730 735Phe Asp Leu
Ile Tyr Pro Ala Asn Leu Glu Arg Phe Asp Pro Asn Phe 740 745 750Thr
Ala Lys Lys Ser Val Leu Ala Ala Asp Asn His Leu Ile Phe Ile 755 760
765Gly Ser Asn Ile Asn Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr
770 775 780Leu Phe Gln His Ala Ile Thr Pro Thr Leu Asn Thr Leu Trp
Ile Asn785 790 795 800Gly Gln Lys Ile Glu Asn Met Pro Tyr Gln Thr
Thr Leu Gln Gln Gly 805 810 815Asp Trp Leu Ile Asp Ser Asn Gly Asn
Gly Tyr Leu
Ile Thr Gln Ala 820 825 830Glu Lys Val Asn Val Ser Arg Gln His Gln
Val Ser Ala Glu Asn Lys 835 840 845Asn Arg Gln Pro Thr Glu Gly Asn
Phe Ser Ser Ala Trp Ile Asp His 850 855 860Ser Thr Arg Pro Lys Asp
Ala Ser Tyr Glu Tyr Met Val Phe Leu Asp865 870 875 880Ala Thr Pro
Glu Lys Met Gly Glu Met Ala Gln Lys Phe Arg Glu Asn 885 890 895Asn
Gly Leu Tyr Gln Val Leu Arg Lys Asp Lys Asp Val His Ile Ile 900 905
910Leu Asp Lys Leu Ser Asn Val Thr Gly Tyr Ala Phe Tyr Gln Pro Ala
915 920 925Ser Ile Glu Asp Lys Trp Ile Lys Lys Val Asn Lys Pro Ala
Ile Val 930 935 940Met Thr His Arg Gln Lys Asp Thr Leu Ile Val Ser
Ala Val Thr Pro945 950 955 960Asp Leu Asn Met Thr Arg Gln Lys Ala
Ala Thr Pro Val Thr Ile Asn 965 970 975Val Thr Ile Asn Gly Lys Trp
Gln Ser Ala Asp Lys Asn Ser Glu Val 980 985 990Lys Tyr Gln Val Ser
Gly Asp Asn Thr Glu Leu Thr Phe Thr Ser Tyr 995 1000 1005Phe Gly
Ile Pro Gln Glu Ile Lys Leu Ser Pro Leu Pro 1010 1015
1020318PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Ala Thr Ser Asn Pro Ala Phe Asp Pro Lys Asn Leu
Met Gln Ser Glu1 5 10 15Ile Tyr421DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 4ctaatgggtt attttgtgca a
21517RNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5gcnacnucna ayccngc 17617DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6gcnacnagya ayccngc 17717RNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7uacguyagnc uyuadau
17817RNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8uacguyucrc uyuadau 17925DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
9catttgatcc taaaaatctg atgca 251018DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
10gcaaccagca atcctgca 181120DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 11taaatttcgg actgcatcag
201253DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 12gca acc agc aat cct gca ttt gat cct aaa
aat ctg atg cag tcc gaa 48Ala Thr Ser Asn Pro Ala Phe Asp Pro Lys
Asn Leu Met Gln Ser Glu1 5 10 15att ta 53Ile Tyr1353DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 13taaatttcgg actgcatcag atttttagga tcaaatgcag
gattgctggt tgc 5314122DNAProteus vulgaris 14gatatcactc aatcattaaa
tttaggcaca acgatgggct atcagcgtta tgacaaattt 60aatgaaggac gcattggttt
cactgttagc cagcgtttct aaggagaaaa ataatgccga 120ta
1221560DNAPhaseolus vulgaris 15atgacaaatt taatgaagga cgcattggtt
tcactgttag ccagcgtttc taaggagaaa 6016400DNAProteus
vulgarisCDS(296)..(400) 16cagactgctt atggcaaatt aaccccctct
cttaatcttc gttattcaaa gatattgcag 60gtgacaatga tatcaatcaa cgccacagcc
ttacctattt taatacaggg ggaagtacct 120ttgatattaa aggaaatacc
gttggtggtg acattattag tgcggaatta ggtgcaaatc 180tcgatatcac
tcaatcatta aatttaggca caacgatggg ctatcagcgt tatgacaaat
240ttaatgaagg acgcattggt ttcactgtta gccagcgttt ctaaggagaa aaata atg
298 Met 1ccg ata ttt cgt ttt act gca ctt gca atg aca ttg ggg cta
tta tca 346Pro Ile Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu
Leu Ser 5 10 15gcg cct tat aac gcg atg gca gcc acc agc aat cct gca
ttt gat cct 394Ala Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala
Phe Asp Pro 20 25 30aaa aat 400Lys Asn 351735PRTProteus vulgaris
17Met Pro Ile Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu Leu1
5 10 15Ser Ala Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe
Asp 20 25 30Pro Lys Asn 35
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