U.S. patent application number 10/915160 was filed with the patent office on 2006-02-09 for cloning and expression of recombinant adhesive protein mefp-2 of the blue mussel, mytilus edulis.
Invention is credited to Francisco F. Roberto, Heather G. Silverman.
Application Number | 20060029996 10/915160 |
Document ID | / |
Family ID | 35734195 |
Filed Date | 2006-02-09 |
United States Patent
Application |
20060029996 |
Kind Code |
A1 |
Silverman; Heather G. ; et
al. |
February 9, 2006 |
CLONING AND EXPRESSION OF RECOMBINANT ADHESIVE PROTEIN MEFP-2 OF
THE BLUE MUSSEL, MYTILUS EDULIS
Abstract
The present invention includes a Mytilus edulis cDNA having a
nucleotide sequence that encodes for the Mytilus edulis foot
protein-2 (Mefp-2), an example of a mollusk foot protein. Mefp-2 is
an integral component of the blue mussels' adhesive protein
complex, which allows the mussel to attach to objects underwater.
The isolation, purification and sequencing of the Mefp-2 gene will
allow researchers to produce Mefp-2 protein using genetic
engineering techniques. The discovery of Mefp-2 gene sequences will
also allow scientists to better understand how the blue mussel
creates its waterproof adhesive protein complex.
Inventors: |
Silverman; Heather G.;
(Idaho Falls, ID) ; Roberto; Francisco F.; (Idaho
Falls, ID) |
Correspondence
Address: |
STEPHEN R. CHRISTIAN
BBWI
PO BOX 1625
IDAHO FALLS
ID
83415-3899
US
|
Family ID: |
35734195 |
Appl. No.: |
10/915160 |
Filed: |
August 9, 2004 |
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/43504 20130101;
C07H 21/04 20130101 |
Class at
Publication: |
435/069.1 ;
435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C07K 14/435 20060101
C07K014/435; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C12N 5/06 20060101 C12N005/06 |
Goverment Interests
U.S. GOVERNMENT RIGHTS
[0002] The United States Government may have rights in this
invention pursuant to Contract No. DE-AC07-99ID13727 between the
U.S. Department of Energy and Bechtel BWXT Idaho, LLC, representing
the Idaho National Engineering & Environmental Laboratory
(INEEL).
Claims
1. (canceled)
2. (canceled)
3. The isolated nucleic acid sequence of claim 25, wherein the
nucleotide sequence comprises the nucleotide sequence of SEQ ID
NO:1, or a degenerate variant of SEQ ID NO:1.
4. The isolated nucleic acid sequence of claim 25, wherein the
nucleotide sequence consists of SEQ ID NO:1.
5. The isolated nucleic acid sequence of claim 25, wherein the
nucleotide sequence comprises at least 500 contiguous nucleotides
of SEQ ID NO:1.
6. (canceled)
7. The isolated nucleic acid sequence of claim 25, wherein the
nucleotide sequence has at least 98% identity to SEQ ID NO:1.
8. The isolated nucleic acid sequence of claim 25, wherein the
nucleotide sequence encodes a polypeptide having the sequence of
SEQ ID NO:2 with a conservative amino acid substitution.
9. (canceled)
10. The isolated nucleic acid sequence of claim 27, wherein the
nucleotide sequence comprises the nucleotide sequence of SEQ ID
NO:3, or a degenerate variant of SEQ ID NO:3.
11. The isolated nucleic acid sequence of claim 27, wherein the
nucleotide sequence consists of SEQ ID NO:3.
12. The isolated nucleic acid sequence of claim 27, wherein the
nucleotide sequence comprises at least 500 contiguous nucleotides
of SEQ ID NO:3.
13. (canceled)
14. The isolated nucleic acid sequence of claim 27, wherein the
nucleotide sequence has at least 98% identity to SEQ ID NO:3.
15. The isolated nucleic acid sequence of claim 27, wherein the
nucleotide sequence comprises a sequence that encodes a polypeptide
having the sequence of SEQ ID NO:4 with a conservative amino acid
substitution.
16. (canceled)
17. The isolated nucleic acid sequence of claim 30, wherein the
nucleotide sequence comprises the nucleotide sequence of SEQ ID
NO:5, or a degenerate variant of SEQ ID NO:5.
18. The isolated nucleic acid sequence of claim 30, wherein the
nucleotide sequence consists of SEQ ID NO:5.
19. The isolated nucleic acid sequence of claim 30, wherein the
nucleotide sequence comprises at least 500 contiguous nucleotides
of SEQ ID NO:5.
20. (canceled)
21. The isolated nucleic acid sequence of claim 130, wherein the
nucleotide sequence has at least 98% identity to SEQ ID NO:5.
22. The isolated nucleic acid sequence of claim 30, wherein the
nucleotide sequence encodes a polypeptide having the sequence of
SEQ ID NO:6 with a conservative amino acid substitution.
23. (canceled)
24. An expression vector comprising the isolated nucleotide seqence
of claim 25, operably linked to an expression control sequence.
25. An isolated nucleic acid sequence encoding an adhesive protein
derived from a mollusk, comprising a nucleotide sequence encoding a
polypeptide having 90% identity to SEQ ID NO:2.
26. The isolated nucleic acid sequence of claim 25, wherein the
nucleotide sequence encodes the polypeptide consisting of SEQ ID
NO:2.
27. An isolated nucleic acid sequence encoding an adhesive protein
derived from a mollusk, comprising a nucleotide sequence encoding a
polypeptide having 90% identity to SEQ ID NO:4.
28. The isolated nucleic acid sequence of claim 27, wherein the
nucleotide sequence encodes the polypeptide consisting of SEQ ID
NO:4.
29. An expression vector comprising the nucleotide sequence of
claim 27, operably linked to an expression control sequence.
30. An isolated nucleic acid sequence encoding an adhesive protein
derived from a mollusk, comprising a nucleotide sequence encoding a
polypeptide having 90% identity to SEQ ID NO:6.
31. The isolated nucleic acid sequence of claim 30, wherein the
nucleotide sequence encodes the polypeptide consisting of SEQ ID
NO:6.
32. An expression vector comprising the nucleotide sequence of
claim 30, operably linked to an expression control sequence.
Description
CROSS REFERENCED APPLICATIONS
[0001] This patent application was filed by Applicants on the same
day as another patent application filed by Applicants entitled
"CLONING AND EXPRESSION OF RECOMBINANT ADHESIVE PROTEIN MEFP-10F
THE BLUE MUSSEL, MYTILUS EDULIS", having Serial No. ______.
TECHNICAL FIELD
[0003] The invention relates to isolated or purified nucleic acid
molecules encoding an adhesive protein, for example, Mefp-2 of the
blue mussel, Mytilus edulis. Adhesives that can be derived from the
present invention can be used in a variety of fields including but
not limited to: military applications, construction products,
plastics, electronics, automobile and aviation products as well as
several biomedical fields.
SEQUENCE LISTINGS
[0004] The electronic readable copy and paper copy of the sequence
listing for this invention are identical.
BACKGROUND OF THE INVENTION
[0005] Mytilus edulis, also termed the common edible mussel or blue
mussel, constitutes most of the world's commercial production of
cultured mussels, along with the closely related species Mytilts
galloprovincialis. Besides their use in food culturing, mussels
(which is an example of a molusk) have also been used to monitor
pollutants in coastal marine waters. The most extensive research
about the adhesive properties of mussels has been with M.
edulis.
[0006] Marine mussels, like the edible blue mussel, M. edulis,
attach to a variety of surfaces in an aqueous environment using a
natural adhesive that is incredibly strong and durable. There are
no conventional glues that can be applied in an aqueous environment
and are impervious to water and turbulent forces. Prior research
has shown that one of the proteins in the adhesive, Mytilus edulis
foot protein 1 (Mefp-1), bonds to glass, plastic, wood, concrete
and Teflon. Nine other adhesive-related proteins from M. edulis
have been identified to date. A tenth is implicated, but has not
been identified. The precise mechanism for assembly of the ten
proteins is not understood (Mefp-1, -2, -3, -4, -5; Collagens:
Precollagen-D, -P (variant P22 and P33), Precollagen-NG, Proximal
Matrix Thread Protein (1 and la); catechol oxidase). There also may
be additional proteins involved in the formation of the
adhesive.
[0007] Individual protein components have been previously
identified from byssal structures through protein isolation and
amino acid analysis, revealing repetitive amino acid motifs and
modified amino acids with unique characteristics not found in other
biological systems. Proposed mechanisms for the strength and
waterproof properties of the adhesive formed, relate to these
recurring amino acid motifs and hydroxylated amino acids found in
many of the protein components. Commercial recombinant protein
products consisting of either the partial amino acid sequence of
Mefp-1 or repeats of the unique decapeptide motif have been
marketed in the past. However, no commercial product incorporates
any of the other proteins known to be involved in underwater
adhesion by the M. edulis mussel. Furthermore, these products are a
result of protein isolation techniques and NOT recombinant DNA
techniques.
[0008] Initial strategies for identifying the adhesive proteins of
the byssus of M. edulis involved purification of the proteins
directly from the byssi of thousands of animals. About 10,000
mussels are needed to produce 1 gram of adhesive. Thus, subsequent
purification and microscopic analysis require(d) the sacrifice of
many mussels. This is neither environmentally friendly nor
economically practical. When the original mussel adhesive protein,
MAP, was identified, only the amino acid motif common to this
protein, also referred to as Mefp-1, (a decapeptide repeat
occurring .about.80 times) was used in an alternate host production
scheme. This MAP recombinant protein did/does have substantial
adhesive properties; however, the (complete) gene sequence for
Mefp-1 and the other proteins involved in byssus formation are
necessary for mimicking the bioadhesive. In addition to a full
length Mefp-1, isolating, purifying and sequencing the DNA sequence
of M. edulis' foot protein-2 (Mefp-2) are critically important and
are objectives of the present invention.
[0009] The mussel byssus is an extracorporeal structure that
consists of a stem, thread, and a plaque (also referred to as a pad
or disc) (See FIG. 1) This exogenous attachment device was first
described in Brown C H, Some Structural Proteins of Mytilus edulis,
Quarterly Journal of Microscopical Science, 93(4): 487 (1952). High
concentrations of polyphenolic proteins (e.g. L-DOPA), the presence
of collagen, and the presence of a catechol oxidase were among the
first observations of byssal attachments. Environmental factors
such as salinity, temperature, pH, season, and substratum choice,
as well as biological factors such as age and metabolic state of
the animal effect the efficiency and strength of
bonding/attachment. See Crisp D J, Walker G, Young G A, Yule A B,
Adhesion and Substrate Choice in Mussels and Barnacles, Journal of
Colloid and Interface Science, 104 (1): 40-50 (1985).
[0010] The stem is rooted in the byssal retractor muscles at the
base of the foot organ. See Crisp D J, Walker G, Young G A, Yule A
B, Adhesion and Substrate Choice in Mussels and Barnacles, Journal
of Colloid and Interface Science, 104 (1): 40-50 (1985). The byssal
threads, flexible structures of variable dimensions (e.g. -0.1 mm
diameter, 2-4 cm length) and strength, originate from the stem. A
byssal thread consists of a flexible, collagenous inner core
surrounded by a hard, browned polyphenolic protein. Numerous
researchers photographed the collagen core in the 1930's (See Brown
C H, Some Structural Proteins of Mytilus edulis, Quarterly Journal
of Microscopical Science, 93(4): 487 (1952))--well before three
unique, collagenous proteins were identified and characterized by
J. H. Waite and colleagues. The outer polyphenolic protein,
believed to undergo a curing or quinone tanning-type reaction with
a specialized catechol/polyphenol oxidase enzyme, is traditionally
designated as Mytilus edulis foot protein 1, Mefp-1, or MAP.
(Designation of the byssal thread polyphenolic adhesive protein, as
well as subsequent adhesive proteins identified in M. edulis, is
preceded by the genus and species: e.g. Mytilus edulis foot protein
1=Mefp-1).
[0011] The breaking energy of byssal threads is reported to be
12.50.times.10.sup.6 Jm.sup.-3, vs tendon (2.times.10.sup.6
Jm.sup.-3 to 5.times.10.sup.6 .mu.m-3) and silk (50.times.10.sup.6
Jm.sup.-3 to 180.times.10.sup.6 Jm.sup.-3; See Denny M W, Biology
and the Mechanics of the Wave Swept Environment, Princeton:
Princeton University Press (1988); Qin X X, Waite J H, Exotic
Collagen Gradients in the Byssus of the Mussel, Mytilus edulis,
Journal of Experimental Biology, 198 (3): 633-644 (1995). Bond
strengths range from 0.1 to 10.times.10.sup.6 Nm .sup.2 depending
on the substratum. (See Waite J H, Reverse Engineering of
Bioadhesion in Marine Mussels, Bioartificial Organs II: Technology,
Medicine, and Materials Annals of the New York Academy of Sciences,
875: 301-309 (1999)). Byssal thread strength at the distal portion
of threads is as strong as vertebrate tendon, but 3-5.times. more
extensible (see, Qin X X, Waite J H, A Potential Mediator of
Collagenous Block Copolymer Gradients in Mussel Byssal Threads,
Proceedings of the National Academy of Sciences of the United
States of America, 95 (18):10517-10522 (1998)). Byssal thread
strength at the proximal portion of threads is weaker, but
15-20.times. more extensible. Strain energy density of threads
approaches that of silk at approximately 6.times. tougher than
tendon. Byssal threads can recover initial length and stiffness
given sufficient relaxation time (See Bell E C, Gosline J M,
Mechanical Design of Mussel Byssus: Material Yield Enhances
Attachment Strength, Journal of Experimental Biology, 199 (4):
1005-1017 (1996). The byssal structure culminates in a polyphasic
plaque of varying size, dependent upon both the size of the animal
and the age of the byssus (See Crisp D J, Walker G, Young G A, Yule
A B, Adhesion and Substrate Choice in Mussels and Barnacles,
Journal of Colloid and Interface Science, 104 (1): 40-50 (1985).
Plaques are commonly only .about.0.15 mm in diameter where they
meet the thread, and .about.2-3 mm diameter at the substrate
interface. Plaque formation occurs from the deposition of proteins
that originate from the foot organ. To date, four specialized
adhesive proteins have been identified in byssal plaques from M.
edulis: Mefp-2, Mefp-3, Mefp-4 and Mefp-5.
[0012] In spite of the extensive research in this area, and
relative success in patenting and commercializing aspects of these
adhesive proteins, a complete understanding of how the byssus is
assembled from its component proteins, and the role each protein
plays in successful assembly and attachment has not been achieved.
A major hurdle has been, and remains, large-scale production of
protein in quantities to allow extensive study outside of the
byssus. This invention describes nucleotide sequences from cDNAs
for Mefp-2 for the first time.
SUMMARY OF THE INVENTION
[0013] One aspect of the invention is an isolated and purified
nucleic acid comprising the nucleotide sequence in (SEQ ID. NO: 1;
a c-DNA sequence) which encodes a biologically active Mefp-2
peptide fragment.
[0014] Another aspect of the invention is an isolated and purified
nucleic acid comprising the nucleotide sequence in (SEQ ID. NO: 3;
a c-DNA sequence) which encodes a biologically active Mefp-2
peptide fragment.
[0015] Another aspect of the invention is an isolated and purified
nucleic acid comprising the nucleotide sequence in (SEQ ID. NO: 5;
a c-DNA sequence) which encodes a biologically active Mefp-2
peptide fragment.
[0016] The invention also relates to methods of using the isolated
and purified DNA sequences to express the polypeptides which they
encode.
[0017] Yet another aspect of the invention is a method of producing
Mefp-2 protein which comprises incorporating the nucleic acids
having the sequences provided by this invention into an expression
vector, transforming a host cell with the vector and culturing the
transformed host cell under conditions which result in expression
of the gene.
[0018] Another aspect of the invention is a nucleic acid sequence
that is capable of hybridizing under stringent conditions to a
nucleotide sequence found in (SEQ ID NO: 1), (SEQ ID NO: 3) or (SEQ
ID NO: 5), or their complements.
[0019] Another aspect of the invention is a nucleic acid molecule
that includes the nucleotide sequence set forth in (SEQ ID NO: 1),
(SEQ ID NO: 3) or (SEQ ID NO: 5), or degenerate variants
thereof.
[0020] Another aspect of the invention is an RNA molecule that
includes the nucleotide sequence set forth in (SEQ ID NO: 1), (SEQ
ID NO: 3) or (SEQ ID NO: 5), or degenerate variants thereof,
wherein Uracil (U) is substituted for Thymine (T).
[0021] Also included in the invention are nucleotides carrying
modifications such as substitutions, small deletions, insertions or
inversions which still encode proteins having substantially the
same activity as the protein of (SEQ ID NO: 2), (SEQ ID NO: 4) or
(SEQ ID NO: 6). Included are nucleic acid molecules having a
sequence which is at least 90% identical to the nucleotide sequence
shown in (SEQ ID NO: 1), (SEQ ID NO: 3) or (SEQ ID NO: 5)
respectively.
[0022] Another aspect of this invention is genetically engineered
polypeptides created using the isolated and purified nucleotide
sequences of this invention.
[0023] Yet another aspect of this invention is utilizing the
genetically engineered polypeptides created using the isolated and
purified nucleotide sequences of this invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a schematic illustration of the byssal structures
of M. edulis adapted from Waite J. H., Chem. Ind. p. 607 (1991) and
Waite J. H, J. Comp. Physiol (B), p. 451 (1986).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0025] In practicing the present invention several conventional
techniques in microbiology and molecular biology (recombinant DNA)
are used. Such techniques are well known and are explained in, for
example, Sambrook, 1999, Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; DNA Cloning: A practical Approach, 1985 (D. N. Glover
ed); Current Protocols in Molecular Biology, John Wiley & Sons,
Inc. (1994) and all more recent editions of these publications.
Definitions
[0026] Before proceeding further with a description of the specific
embodiments of the present invention, a number of terms will be
defined.
[0027] As used herein, a compound or molecule is an organic or
inorganic assembly of atoms of any size, and can include
macromolecules, peptides, polypeptides, whole proteins, and
polynucleotides.
[0028] As used herein, a polynucleotide is a nucleic acid of more
than one nucleotide. A polynucleotide can be made up of multiple
poly-nucleotide units that are referred to be a description of the
unit. For example, a polynucleotide can comprise within its bounds
a polynucleotide(s) having a coding sequence(s), a
polynucleotide(s) that is a regulatory region(s) and/or other
polynucleotide units commonly used in the art.
[0029] The isolated nucleic acid molecule of the present invention
can include a deoxyribonucleic acid molecule (DNA), such as genomic
DNA and complementary cDNA which can be single (coding or noncoding
strand) or double stranded, as well as synthetic DNA, such as
synthesized single stranded polynucleotide. The isolated nucleic
acid molecule of the present invention can also include a
ribonucleic acid molecule (RNA).
[0030] The determination of percent identity or homology between
two sequences is accomplished using the algorithm of Karlin and
Altschul (1990) Proc. Nat'l Acad. Sci. USA 87: 2264-2268, modified
as in Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA
90:5873-5877. Such an algorithm is incorporated into the NBLAST and
XBLAST programs of Altschul et al. (1990) J. Mol. Biol.
215:403-410. BLAST nucleotide searches are performed with the
NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to the nucleic acid molecules of the
invention. BLAST protein searches are performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the protein molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST is utilized
as described in Altschul et al. (1997) Nucleic Acids Res. 25:
3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (e.g., XBLAST and
NBLAST) are used. See the website for the national center for
biological information.
[0031] As used herein, the terms hybridization (hybridizing) and
specificity (specific for) in the context of nucleotide sequences
are used interchangeably. The ability of two nucleotide sequences
to hybridize to each other is based upon a degree of
complementarity of the two nucleotide sequences, which in turn is
based on the fraction of matched complementary nucleotide pairs.
The more nucleotides in a given sequence that are complementary to
another sequence, the greater the degree of hybridization of one to
the other. The degree of hybridization also depends on the
conditions of stringency, which include: temperature, solvent
ratios, salt concentrations, and the like.
[0032] In particular, selective hybridization pertains to
conditions in which the degree of hybridization of a polynucleotide
of the invention to its target would require complete or nearly
complete complementarity. The complementarity must be sufficiently
high as to assure that the polynucleotide of the invention will
bind specifically to the target relative to binding other nucleic
acids present in the hybridization medium. With selective
hybridization, complementarity will be 90-100%, preferably 95-100%,
more preferably 100%.
[0033] The term stringent conditions is known in the art from
standard protocols (e.g. Current Protocols in Molecular Biology,
editors F. Ausubel et al., John Wiley and Sons, Inc. 1994) and is
when hydridization to a filter-bound DNA in 0.5M NaHPO.sub.4
(pH7.2), 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65.degree.
C., and washing in 0.1.times.SSC/0.1% SDS at +68.degree. C. is
performed.
[0034] Degenerate variant is the redundancy or degeneracy of the
genetic code as is well known in the art. Thus the nucleic acid
sequences shown in the sequence listing provided only examples
within a larger group of nucleic acids sequences that encode for
the polypeptide desired.
[0035] Isolated nucleic acid will be nucleic acid that is
identified and separated from contaminant nucleic acid encoding
other polypeptides from the source of nucleic acid. The nucleic
acid may be labeled for diagnostic and probe purposes, using any
label known and described in the art as useful in connection with
diagnostic assays.
[0036] Because the genetic code is degenerate, more than one codon
may be used to encode a particular amino acid, and therefore, the
amino acid sequence can be encoded by any set of similar DNA
oligonucleotides. With respect to nucleotides, therefore, the term
derivative(s) is also intended to encompass those DNA sequences
that contain alternative codons which code for the eventual
translation of the identical amino acid.
[0037] Mussel adhesive proteins are scleroproteins--proteins
contributing mechanical strength to supporting structures in
animals. Familiar scleroproteins include collagen, silk, elastin,
fibroin, keratin, fibrin and resilin. Quinone tanning requires a
catecholic precursor (such as catechol oxidase) and the presence of
tanned scleroproteins. See, Waite J H, The Phylogeny and Chemical
Diversity of Quinone-tanned Glues and Varnishes, Comp Biochem
Physiol B., 97(1):19-29 (1990). Individual adhesive proteins from
mussels are derived from the foot organ of the animals. The
proteins are stockpiled in the foot, and then secreted or released
into the environment to form strong attachments underwater. The
proteins involved in adhesion of mussels contain
peptidyl-3-4,-dihydroxy-phenylalanine (DOPA), a constituent not
found in barnacle cement proteins. The reactive, oxidized form of
DOPA, quinone, is thought to provide the moisture-resistance
characteristic of mussel underwater adhesion. See, Yu M, Hwang, J,
Deming, T J, Role of L-3,4-Dihydroxyphenylalanine in Mussel
Adhesive Proteins, Journal of the American Chemical Society, 121:
5825-5826 (1999). DOPA can complex with metal ions and oxides and
semi-metals such as silicone, thus explaining the ability to adhere
to rocks and glass. Other constituents of mussel adhesive proteins
include lysine and glycine. Lysine may contribute to adhesion via
ionic bonding to negatively charged surfaces like collagen and
acidic polysaccharides. Proposed mechanisms for the strength and
waterproof properties of the adhesive formed relate to recurring
amino acid motifs (decapeptide repeats of 75-80 times in Mefp-1)
and the hydroxylated amino acids found in the adhesive proteins.
Polyphenolic proteins are non-toxic, biodegradable and have a low
immunogenicity.
[0038] Mefp-1 is a polyphenolic protein with primarily structural
properties. It is oxidized and cross-linked through the action of a
catechol oxidase to form a hardened sheath (the byssal thread) that
extends between the foot organ of the animal and the surface of
attachment. The inner core of this structure is comprised of four
collagens (with variants) with distinctive domains not found in
other biological systems. This combination of proteins functions
much like a natural epoxy adhesive. The cystine-rich Mefp-2 forms
the foam-like plaque component of the byssus. Mefp-4 and Mefp-5 are
additional proteins located in the plaque. A
hydroxyarginine-containing protein, Mefp-3, is believed to serve as
a primer-like protein for this byssal plaque.
Byssal Plaque Polyphenolic Protein: Mefp-2
[0039] Mefp-2 is found exclusively in byssal plaques, constituting
from 25-40% of the total plaque proteins. Unlike Mefp-1, Mefp-2 is
a smaller adhesive protein (molecular mass 42-47 kDa) with only 2-3
mol % DOPA and no hydroxylation of proline to
trans-2,3-cis-3,4-dihydroxyproline or trans-4-hydroxy-L-proline.
The DOPA residues occur primarily in the N- and C-terminal regions
of the protein. Mefp-2 contains considerable secondary structure
and is relatively resistant to a variety of proteases (compared to
Mefp-1). The high cysteine content (6-7 mol %) coupled with
tandemly repetitive motifs similar to epidermal growth factor,
represents an adhesive protein with a stabilization role in the
byssus (See Inoue K, Takeuchi Y, Miki D, Odo S, Mussel Adhesive
Plaque Protein Gene is a Novel Member of Epidermal Growth
Factor-like Gene Family, Journal of Biological Chemistry, 270 (12):
6698-6701 (1995).
[0040] An Mefp-2 multi-gene family may exist, based on evidence
that at least three different repetitive motifs have been
identified in the primary protein sequence (See Rzepecki L M,
Hansen K M, Waite J H, Characterization of Cysteine-rich
Polyphenolic Protein Family from the Blue Mussel, Mytilus edulis-L,
Biological Bulletin, 183 (1): 123-137 (1992). A published
full-length gene sequence for Mefp-2 has not been available until
now.
Other Byssal Proteins: Mefp-1, Mefp-3, Mefp-4 and Mefp-5
[0041] Mefp-1
[0042] Mefp-1 was the first polyphenolic protein to be identified
in the mussel byssus (See Waite J H, Tanzer M L, Polyphenolic
Substance of Mytilus edulis Novel Adhesive Containing L-Dopa and
Hydroxyproline, Science, 212 (4498): 1038-1040 (1981). The primary
location of Mefp-1 is in the byssal threads, cross-linked via a
polyphenol oxidase to form a schlerotonized sheath around the
flexible, collagen inner-core. Byssal plaques contain approximately
5% of Mefp-1 as well. Mefp-1 adhesive properties are comparable to
synthetic cyanoacrylate and epoxy resins.
[0043] Mefp-1 is a large, basic protein with very little secondary
structure and a molecular mass of 130 kDa. Decapeptide and
hexapeptide repeats containing numerous post-translational
modifications (-60-70% of the amino acid residues are hydroxylated)
provided the first indication of an adhesive-related protein unlike
any others identified in nature. The hexapeptide repeat is AKPTYK
(SEQ ID NO: 22). The major decapeptide consensus repeat, consisting
of AKPSYPPTYK (SEQ ID NO: 23) (where Y represents
3,4-dihydroxyphenyl-alanine (DOPA), "P" represents
trans-2,3-cis-3,4-dihydroxyproline, and P represents
trans-4-hydroxy-L-proline) occurs approximately eighty times in
Mefp-1. DOPA residues constitute 10-15% of the protein (See Waite J
H, Evidence for a Repeating 3,4-Dihydroxyphenylalanine-Containing
and Hydroxyproline-Containing Decapeptide in the Adhesive Protein
of the Mussel, Mytilus edulis, Journal of Biological Chemistry, 258
(5): 2911-2915 (1983). The open conformation of the protein is
believed to allow functional groups full accessibility for
interactions with other proteins and a variety of surfaces,
including glass, Teflon, and metals.
[0044] Mefp-1 has been previously commercialized as a source for
mussel adhesive protein. Companies supplying Mefp-1 have obtained
the pure protein from the byssal structures using protein
extraction techniques (e.g., Sigma-Aldrich; BD Biosciences
Clontech, formerly marketed by BioPolymers Corp of Farmington,
Conn., under the trademark CELL-TAK.RTM.) and recombinant protein
techniques using synthetic gene constructs. However, currently
there are no commercial sources for Mefp-1, due to the high cost of
extraction methods and inconsistencies in quality of protein from
recombinant protein techniques. All of the laboratory-prepared
products were not as strong as the natural protein.
[0045] Mefp-1 requires oxidization by catechol oxidase or
tyrosinase enzymes (or periodontate) in order to render the
tyrosine residues converted to reactive DOPA residues required for
strong adhesion. The enzyme oxidation may serve as an oxidative
agent and as a copolymer. Molecular oxygen can also be used to
oxidize DOPA to a quinone. Possible cross-linking agents are
oxygen, polyvalent metal ions, Fe.sup.3+ and Al.sup.3+, aldehydes
and many types of bi/polyfunctional cross-linkers. The addition of
other macromolecules to the Mefp-1 protein--such as collagen,
casein or keratin--has been recommended by companies in order to
increase the adhesive properties of the individual protein.
[0046] Mefp-1 in the form of CELL-TAK (BioPolymers Corp of
Farmington, Conn.) has been tested as a surgical adhesive between a
number of different cells or tissues from a range of species. For
example, studies testing the efficiency of CELL-TAK (BioPolymers
Corp of Farmington, Conn.) compared to other adhesives have
included porcine cartilage, bone and skin (see, Chivers R A,
Wolowacz R G, The Strength of Adhesive-Bonded Tissue Joints,
International Journal of Adhesion and Adhesives, 17 (2): 127-132
(1997)), rat tissue (see, Schmidt S P, Resser J R, Sims R L,
Mullins D L, Smith D J, The Combined Effects of
Glycyl-L-Histidyl-L-Lysine-Copper (II) and CELL-TAK.RTM.
(BioPolymers Corp of Farmington, Conn.) on the Healing of Linear
Incision Wounds, Wounds A Compendium of Clinical Research and
Practice, 6 (2):62-67 (1994)), rabbit corneas (see, Robin J B,
Picciano P, Kusleika R S, Salazar J, Benedict C, Preliminary
Evaluation of the Use of Mussel Adhesive Protein in Experimental
Epikeratoplasty, Archives of Ophthalmology, 106 (7):973-977
(1988)), and chicken osteoblasts and cartilage cells (see,
Fulkerson J P, Norton L A, Gronowicz G, Picciano P, Massicotte J M,
Nissen C W, Attachment of Epiphyseal Cartilage Cells and 17/28 Rat
Osterosarcoma Osteoblasts using Mussel Adhesive Protein, Journal of
Orthopaedic Research, 8 (6): 793-798 (1990)). Studies have also
included human breast cancer cells and mouse sperm cells. The best
adhesion with CELL-TAK (BioPolymers Corp of Farmington, Conn.) has
been shown to occur with cell cultures. Other testing of CELL-TAK
for industrial applications has included it's use as an enzyme
immobilization matrix in the fabrication of enzyme-based electrodes
(See Saby C, Luong J H T, Mytilus edulis Adhesive Protein (MAP) as
an Enzyme Immobilization Matrix in the Fabrication of Enzyme-Based
Electrodes, Electroanalysis, 10 (17): 1193-1199 (1998)).
[0047] Purified polyphenolic protein was also shown to effectively
immobilize human chorionic gonadotrophin to wells of a microtiter
plate (See Burzio V A, Silva T, Pardo J, Burzio L O, Mussel
Adhesive Enhances the Immobilization of Human Chorionic
Gonadotrophin to a Solid Support, Analytical Biochemistry, 241 (2):
190-194 (1996). In addition, the immunoreactivity of the attached
antigen used in the study was stable for several months. This
example shows a possible tool for polyphenolic proteins in basic
research and medical diagnostics.
[0048] Other Mytilus mussel species contain a protein analogous to
Mefp-1, with differences in the decapeptide repeat frequency,
residue composition, and non-repetitive regions. To date, analogous
proteins to Mgfp-2 have not been reported other than the
identification of the Mefp-2 variants described herein.
[0049] Mefp-3
[0050] Mefp-3 is the smallest byssal adhesive protein identified to
date, with a molecular mass of .about.5-7 kDa. See Papov V V,
Diamond T V, Biemann K, Waite J H, Hydroxyarginine-Containing
Polyphenolic Proteins in the Adhesive Plaques of the Marine Mussel,
Mytilus edulis, Journal of Biological Chemistry, 270 (34):
20183-20192 (1995); Inoue K, Takeuchi Y, Miki D, Odo S, Harayama S,
Waite J H, Cloning, Sequencing and Sites of Expression of Genes for
the Hydroxyarginine-Containing Adhesive-Plaque Protein of the
Mussel, Mytilus galloprovincialis, European Journal of
Biochemistry, 239 (1): 172-176 (1996); Warner S C, Waite J H,
Expression of Multiple Forms of an Adhesive Plaque Protein in an
Individual Mussel, Mytilus edulis, Marine Biology, 134 (4): 729-734
(1999). Mefp-3 contains no repeats, 20-25 mol % DOPA, and a
prevalence of 4-hydroxyarginine and tryptophan residues. Warner S
C, Waite J H, "Expression of Multiple Forms of an Adhesive Plaque
Protein in an Individual Mussel, Mytilus edulis", Marine Biology,
134 (4): 729-734 (1999) identified twenty gene variants (-0.3 kB)
of Mefp-3 in the foot organ; however, only four or five proteins
have actually been detected in plaques deposited on glass or
plastic. The presence of a gene family for Mefp-3 supports the
primer-like function of the protein in adhering to substrata. One
hypothesis has been that deposition of a specific Mefp-3 variant is
dependent upon the surface used for attachment. However, protein
expression specific to substrate attachment has not been
demonstrated to date.
[0051] Mefp-4
[0052] Mefp-4 is another protein identified in byssal plaques, with
a molecular mass of 79 kDa (See, Warner S C, Waite J H, "Expression
of Multiple Forms of an Adhesive Plaque Protein in an Individual
Mussel, Mytilus edulis", Marine Biology, 134 (4): 729-734 (1999);
Vreeland V, Waite J H, Epstein L, "Polyphenols and Oxidases in
Substratum Adhesion by Marine Algae and Mussels", Journal of
Phycology, 34 (1): 1-8 (1998); Weaver, JK, "Isolation,
Purification, and Partial Characterization of a Mussel Byssal
Precursor Protein, Mytilus edulis foot protein 4", MS thesis,
University of Delaware, Newark, (1998)
[0053] Mefp-4 contains elevated levels of glycine, arginine, and
histidine, as well as 4 mol % DOPA. A unique tyrosine-rich
octapeptide is present, with variations in residue substitutions
giving rise to a family of proteins. This very large protein most
likely serves a stabilization role in byssal plaques, as does
Mefp-2. A gene sequence for Mefp-4 has not been identified, nor are
any analogs/homologs from other mussel species available to
date.
[0054] Mefp-5
[0055] Mefp-5 is the most recent identified adhesive-related byssal
plaque protein. See, Waite J H, Qin X X, "Polyphosphoprotein from
the Adhesive Pads of Mytilus edulis", Biochemistry, 40 (9):
2887-2893 (2001). Mefp-5 is a relatively small protein with a
molecular mass of 9.5 kDa, a 27 mol % DOPA content, and the
presence of phosphoserine. Phosphoserine is known to occur in
acidic mineral-binding motifs of proteins that bind calcareous
materials (e.g. osteopontin); therefore, its presence in byssal
plaques may aid in adhesion of one animal to a neighboring mussel's
shell. Mefp-5 was formerly associated with the Mefp-3 family of
variants, and similarly, plays an interfacial role as a primer for
substrate adhesion. See, also M J Sever, et al., Metal-mediated
cross-linking in the generation of a marin-mussel adhesive.
Angewandte Chemie 43(4), 448-450.
[0056] An underwater adhesive will be a valuable asset to the
military and industries such as forest products (composite wood
products), building/construction, plastics, electronics,
automotive, aviation, and the biomedical fields (dentistry,
surgery, orthopedics, ophthalmology). All can benefit from an
environmentally safe, strong, inexpensive alternative to the
conventional adhesives available today. There are no conventional
glues that can be applied in an aqueous environment and are
impervious to water and turbulent forces. The development of a
biomimetic glue product (an adhesive that employs man-made
materials to mimic the efficient attachment mechanisms of the
natural mussel) will revolutionize the field of adhesive
technology. Mussel adhesive proteins represent a tantalizing target
in the field of biomimetics. The challenge of resisting the effects
of water: (i) its ability through hydrogen bonding to interfere
with initial bonding between the substrate and adhesive; (ii) the
attack by water on the adhesive-substrate interface through wicking
and crazing; (iii) swelling of adhesive (and failure of the bond
junction) through water absorption; and (iv) dissolution or erosion
of the adhesive, have been met by the mussel byssus and the protein
constituents secreted during its synthesis. For more than 20 years,
researchers have studied mussel adhesion to gain clues to design
better glues for wet environments, such as in dentistry, as a
surgical glue and in industry. Two commercial products for
attachment of cells to plastic vessels in cell culture applications
have been introduced (Cell-Tak/BioPolymers, Inc., AdheraCell/Genex
Corp.), and several U.S. patents cover aspects of the repeating
decapeptide motif, isolation of polyphenolic proteins from mussels
and recombinant forms of Mefp-1.
Genetic Approaches
[0057] Reverse-genetics approaches to obtaining complete gene
sequences, enzymatic screening of a cDNA library from the foot
organ of M. edulis, and the use of DNA probes allow for detection
of transcripts actively expressed and transcribed by the mussel.
With the complete gene sequences, an alternate host system can be
employed to produce the adhesive proteins of interest for future
analyses from protein chemistry, novel microscopy, and adhesive
science disciplines. The adhesives industry will require a large
quantity of protein to perform adequate testing and analyses for
future adhesive technologies.
[0058] In the first reverse-genetics strategy, an approach is taken
to identify known genes for adhesive proteins of interest. For this
method, PCR (polymerase chain reaction) primers are designed for
the genes of interest based on available nucleotide and amino acid
sequences from M. edulis and other mussel species. The primers are
combined with total RNA isolated from the foot organ of M. edulis
in an RT-PCR (reverse transcription followed by PCR) reaction to
yield a product corresponding to the gene of interest. This cDNA
(c="complementary") product is then inserted (cloned) into a
plasmid vector (currently obtained from a vendor). The clone for
the adhesive gene of interest is now packaged for analysis by DNA
sequencing and for insertion (transformation) into a suitable host
for recombinant protein expression. DNA sequencing of the clone is
critical in 1) determining that the clone is full-length e.g.
contains the start and stop signal for translation of the full gene
to protein, and 2) identifying variants in any of the gene
sequences.
[0059] In the second reverse-genetics approach, a cDNA library is
constructed from RNA isolated from the foot organ of M. edulis.
This library consists of individual clones in wells of a microtiter
plate. High-throughput DNA sequencing of the microtiter plates
containing the clones, followed by analysis using available
bioinformatics software programs, will enable 1) a determination of
all of the genes presently expressed in the foot of the mussel, and
2) a determination of known and possibly novel adhesive proteins
expressed in the foot of the mussel. The treatment of mussels prior
to excision of their foot organ for RNA isolation (e.g. exposure to
various surfaces, water conditions) may play a role in the
expression of genes in the foot organ.
[0060] An enzymatic assay is a third strategy to obtain the
polyphenoloxidase (catechol oxidase) gene. In this assay,
microtiter plates containing either 1) all clones from a foot organ
cDNA library or 2) only clones identified by DNA sequencing to
resemble a polyphenoloxidase enzyme, are subjected to addition of
an appropriate substrate for colorimetric indication of active
enzyme activity. It is important that the active form of the
protein be determined for subsequent adhesive formulation
determinations.
[0061] A fourth strategy to obtain genes for adhesive proteins
involves the development of nucleotide probes based on known DNA
sequences or protein sequence motifs in the respective genes. These
probes are then tested against a cDNA foot library from M.
edulis.
Preferred Embodiments
[0062] The present invention relates to the adhesive protein,
Mepf-2 and the nucleotide sequences encoding such protein, found in
the blue mussel, Mytilus edulis. Sequence ID NO: 1, SEQ ID NO: 3
and SEQ ID NO: 5 describe the DNA sequence encoding Mepf-2 (SEQ ID
NOs: 1, 2, and 3 are representative of the coding sequence, since
they were generated from c-DNAs). (Sequence ID NO: 2), (SEQ ID NO:
4) and (SEQ ID NO: 6) illustrate the corresponding amino acid
sequences for the abovementioned nucleotide sequences.
Nucleotide Sequences
[0063] The scope of the present invention is not limited to the
exact sequence of the nucleotide sequences set forth in (SEQ ID NO:
1), (SEQ ID NO: 3) and (SEQ ID NO: 5) or the use thereof. The
invention contemplates certain modifications to the sequence,
including deletions, insertions, and substitutions, that are well
known to those skilled in the art. For example, the invention
contemplates modifications to the sequence found in (SEQ ID NO:1),
(SEQ ID NO: 3) and (SEQ ID NO: 5) with codons that encode amino
acids that are chemically equivalent to the amino acids in the
native protein. An amino acid substitution involving the
substitution of amino acid with a chemically equivalent amino acid
includes a conserved amino acid substitution.
[0064] Chemical equivalency can be determined by one or more the
following characteristics: charge, size,
hydrophobicity/hydrophilicity, cyclic/non-cyclic,
aromatic/non-aromatic etc. For example, a codon encoding a neutral
non-polar amino acid can be substituted with another codon that
encodes a neutral non-polar amino acid, with a reasonable
expectation of producing a biologically equivalent protein.
[0065] Amino acids can generally be classified into four groups.
Acidic residues are hydrophilic and have a negative charge to loss
of H.sup.+ at physiological pH. Basic residues are also hydrophilic
but have a positive charge to association with H.sup.+ at
physiological pH. Neutral nonpolar residues are hydrophobic and are
not charged at physiological pH. Neutral polar residues are
hydrophilic and are not charged at physiological pH. Amino acid
residues can be further classified as cyclic or noncyclic and
aromatic or nonaromatic, self-explanatory classifications with
respect to side chain substituent groups of the residues, and as
small or large. The residue is considered small if it contains a
total of 4 carbon atoms or less, inclusive of the carboxylcarbon.
Small residues are always non-aromatic.
[0066] Of naturally occurring amino acids, aspartic acid and
glutamic acid are acidic; arginine and lysine are basic and
noncylclic; histidine is basic and cyclic; glycine, serine and
cysteine are neutral, polar and small; alanine is neutral, nonpolar
and small; threonine, asparagine and glutamine are neutral, polar,
large and nonaromatic; tyrosine is neutral, polar, large and
aromatic; valine, isoleucine, leucine and methionine are neutral,
nonpolar, large and nonaromatic; and phenylalanine and tryptophan
are neutral, nonpolar, large and aromatic. Proline, although
technically neutral, nonpolar, large, cyclic and nonaromatic is a
special case due to its known effects on secondary conformation of
peptide chains, and is not, therefore included in this defined
group.
[0067] There are also common amino acids which are not encoded by
the genetic code include by example and not limitation: sarcosine,
beta-alanine, 2,3-diamino propionic and alpha-aminisobutryric acid
which are neutral, nonpolar and small; t-butylalanine,
t-butylglycine, methylisoleucine, norleucine and cyclohexylalanine
which are neutral, nonpolar, large and nonaromatic; ornithine which
is basic and non-cylclic; cysteic acid which is acidic; citrulline,
acetyl lysine and methionine sulfoxide which are neutral, polar,
large and nonaromatic; and phenylglycine, 2-naphtylalanine,
.beta.-2-thienylalanine and
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid which are neutral,
nonpolar, large and aromatic. Other modifications are known in the
art some of which are discussed in U.S. Pat. No. 6,465,237 issued
to Tomlinson on Oct. 15, 2002.
Cloning and Sequencing of cDNA Encoding Mepf-2: SEQ ID NO: 1, 3,
and 5
[0068] For SEQ ID NO: 5 (clone designation QTB10): Total RNA from
the foot organ of M. edulis was supplied to the customer by
Invitrogen Corporation (Carlsbad, Calif.). Invitrogen's cDNA
library was constructed using the following strategy. First strand
cDNA was synthesized using AMV Reverse Transcriptase with a Not T
primer. The Not I primer is a 39 base pair primer which consists of
18 T residues and a Not I restriction site. The RNA-cDNA hybrid
created by first strand synthesis was converted to double stranded
cDNA by DNA Polymerase I in combination with RNase H and E. coli
DNA ligase. After addition of BstX I adapters, the cDNA was
digested with Not I and sized on an agarose gel. Size selected cDNA
(>500 bp) was ligated into BstX I/Not I digested phagemid vector
pYES2 and transformed into the E. coli strain TOP10F'. pYES2 is a
yeast expression vector. Library amplification was performed by
plating over 20 large plates and incubating overnight at 37.degree.
C. The cells were scraped from the plates, resuspended into SOC
media/20% glycerol and aliquoted into 6 tubes with each vial
containing approximately 2 mL. Vials were stored at 80.degree. C.
until use. Validation: number of primary
recombinants=4.35.times.10.sup.6 ratio containing inserts=10/10,
average insert size of the clones analyzed=1.22 kB. The original
cDNA library from Invitrogen was designated as #1 (I). Subsequent
replications and platings were designated as cDNA libraries #2 (II)
and #3 (III). TABLE-US-00001 TABLE 1 Primers for RT-PCR: Mefp-2
(SEQ ID NO: 1 and 3) Restriction Primer F/R Target DNA Sequence: 5'
to 3' nt Site Amino Acids 514 F Mefp-2 gcggccgccacagaagcatcatgttgt
(SEQ ID NO: 17) 31 Not I . . . MLFS (SEQ ID NO: 20) tttc 515 R
Mefp-2 gagctcgtctaggttaacttaatactc (SEQ ID NO: 18) 30 Sac I . . .
DEY* (SEQ ID NO: 21) gtc
[0069] For SEQ ID NO: 1 and 3: Specific primers were designed (as
shown in Table 1) based upon GenBank Accession No. D43794, Mytilus
galloprovincialis (SEQ ID NO: 19) mRNA for adhesive plaque matrix
protein. Primer set 514(SEQ ID NO: 17)/515(SEQ ID NO: 18) produced
clones #2 (SEQ ID NO: 1) and #7 (SEQ ID NO: 3) The M.
galloprovincialis sequence was used because it was assumed an
analogous protein is present in M. edulis.
[0070] The nucleotide sequence for Mepf-2 clone #2 (SEQ ID NO: 1)
and clone # 7 (SEQ ID NO: 3) was amplified by RT-PCR using the
designed primers with total RNA isolated from the foot organ of M.
edulis. Following first strand cDNA synthesis, PCR was carried out
as described for clone #2: 1 .mu.L cDNA, 5 .mu.L 10.times. Buffer
for Accuzyme DNA Polymerase (for a 2 mM MgCl.sub.2 final
concentration; Bioline; Randolph, Mass.), 1 .mu.L dNTPs (at 10 mM
each), 2 .mu.L primer set 514 (SEQ ID NO: 17)/515(SEQ ID NO:18)
(for Mefp-2; at 100 pmol/.mu.L), 40 .mu.L sterile water and 1.0 KLL
Accuzyme DNA Polymerase were added to a thin-walled 0.5 mL PCR
tube. Amplification was performed on a PerkinElmer 9700
thermocycler (PerkinElmer, Inc.; Boston, Mass.) under the following
conditions: 95.degree. C.-3 minutes; 30 cycles of: 95.degree. C.-30
seconds, 50.degree. C.-1:00 minute, 72.degree. C.-2:00 minutes;
72.degree. C.-10:00 minutes; final hold at 4.degree. C. The PCR
reaction was analyzed on a 1% agarose gel.
[0071] Following first strand cDNA synthesis, PCR was carried out
as described for clone #7: 1 .mu.L cDNA, 5 .mu.L 10.times. Buffer
for Taq PCR buffer (Promega; Madison, Wis.), 1 .mu.L dNTPs (at 10
mM each), 2 .mu.L primer set 514 (SEQ ID NO: 17)/515(SEQ ID NO:18)
(for Mefp-2; at 100 pmol/.mu.L), 37.5 .mu.L sterile water and 0.5
.mu.L Taq DNA Polymerase were added to a thin-walled 0.5 mL PCR
tube. Amplification was performed on a Perkin-Elmer 9700
thermocycler as described above for clone #2. The PCR reaction was
analyzed on a 1% agarose gel.
[0072] Cloning was performed per the pYES2.1 TOPO TA Cloning Kit
(Invitrogen; Carlsbad, Calif.). Transformants were picked and
screened by restriction enzyme digestion (Sacd and NotI double
restriction digest, per New England BioLabs; Beverly, Mass.) and
DNA sequencing. Clone designations #2 and #7 (SEQ ID NOs:1 and 3,
respectively) were determined to be complete cDNA clones for
Mefp-2.
[0073] DNA sequencing of all potential Mefp-2 clones was performed
with a LiCor 4000L DNA Sequencer (LiCor Inc.; Lincoln, Nebr.) and
with an ABI 3700 DNA Sequencer using BigDye v2.0 and v3.0
chemistries (Applied Biosystems; Foster City, Calif.).
Oligonucleotide sequencing primers were obtained from Invitrogen
cloning kits, LiCor, Operon Technologies, Inc. (Alameda, Calif.),
and MWG Biotech (UK). Primers used with the LiCor sequencer were
IRD 800 dye-labeled. Primers used with the Applied Biosystems
sequencer were un-labeled. See Table 2 for details of DNA
sequencing primers used.
[0074] Screening of the cDNA libraries was performed following
96-well plasmid preparation methods from various vendors (e.g.
Qiagen (Alameda, Calif.) and Promega (Madison, Wis.)).
[0075] Sequencing primers designed for vector targets were designed
based upon vector sequences provided by Invitrogen. Primers
designed by the inventors for targeting DNA sequence of Mefp-2 were
based upon clone #2, #7 and QTB10 consensus sequences.
[0076] Sequencing primers for vector targets were obtained from
Invitrogen. Primers designed by inventors (labeled HS/FFR) for
targeting the DNA sequence of Mefp-2 were based upon clone #2, #7
and QTB10 consensus sequence (SEQ ID NO: 1, 3, and 5).
TABLE-US-00002 TABLE 2 Sequencing Primers Target: F/ Vector or DNA
Primer R Sequence DNA Sequence: 5' to 3' nt T7 F pYES2
TAATACGACTCACTATAGGG (SEQ ID NO: 7) 20 (Standard) and Invitrogen
pYES2.1/V5-His- Corporation TOPO V5C-term R pYES2.1/V5-His-
ACCGAGGAGAGGGTTAGGGAT (SEQ ID NO: 8) 21 Reverse TOPO Invitrogen
Corporation 506 R pYES2 TTTCGGTTAGAGCGGATG (SEQ ID NO: 9) 18 by
HS/FFR 507 R pYES2 AGGGCGTGAATGTAAGCGTG (SEQ ID NO: 10) 20 by
HS/FFR 508 F Mefp-2 internal TTTGGTCCAGAATGCGAG (SEQ ID NO: 11) 18
by HS/FFR Sq1 F Mefp-2 internal CTTTGGCAGACTTTGCG (SEQ ID NO: 12)
17 by HS/FFR Sq2 F Mefp-2 internal ACGGAAAGTGCTCACCC (SEQ ID ON:
13) 17 by HS/FFR Sq3 F Mefp-2 internal AAGTGCTCACCCTTGGG (SEQ ID
NO: 14) 17 by HS/FFR FP-1 F pPDM-1 CCCAATACGCAAACCGCCTCT (SEQ ID
NO: 15) 21 EpiCentre RP-1 R pPDM-1 TTAGAAAAATAAACAAATAGGGG (SEQ ID
NO: 16) 25 EpiCentre TT
[0077] It should be noted that Epicentre FP-1 (SEQ ID NO: 15) and
RP-1 (SEQ ID NO: 16) were used for Mefp-1 and not used for
Mefp-2.
Expression of Mefp-2 Protein
[0078] Expression of recombinant Mefp-2 protein from clone #2, #7
and QTB10 (SEQ ID NO: 1, 3, and 5) is performed by following the
protocol set forth by Invitrogen. Expression is performed with the
pYES2 system in the yeast strain Saccharomyces cerevisiae. A
30-liter fermentor (Bio Flo 4500--New Brunswick Scientific; Edison,
New Jersey) is used to scale-up from the Invitrogen protocol.
[0079] Having described the basic concept of the invention, it will
be apparent to those skilled in the art that the foregoing detailed
disclosure is intended to be presented by way of example only, and
is not limiting. Various alterations, improvements, and
modifications are intended to be suggested and are within the scope
and spirit of the present invention. Additionally, the recited
order of the elements or sequences, or the use of numbers, letters
or other designations therefore, is not intended to limit the
claimed processes to any order except as may be specified in the
claims. Accordingly, the invention is limited only by the following
claims and equivalents thereto.
[0080] All publications and patent documents cited in this
application are incorporated by reference in their entirety for all
purposes to the same extent as if each individual publication or
patent document were so individually denoted.
Sequence CWU 1
1
23 1 1527 DNA Mytilus edulis 1 atgttgtttt ctttctttct tttgcttact
tgtacccagc tttgcctagg aacgtcacca 60 cgtccatatg atgatgatga
ggatgactac acaccaccag tatacaagcc ttcaccatcg 120 agatatcgac
cagtaaaccc ctgtttaaag aagccatgta aatacaatgg agtatgtaaa 180
cccagtggtg gctcctacaa atgtgtctgc aaaggaggat actatggata caattgcaac
240 ctaaaaaacg catgtaaacc aaaccaatgt aaaaataaaa gtagatgtat
acctgttgga 300 aaaacattta aatgtgaatg tagaaatgga aactttggca
gactttgcga aagaaatgta 360 tgtagcccta atccttgtaa gaacaaagga
aagtgcgccc ccttgggaaa gacaggatat 420 aaatgtacat gtagtggagg
atacactggt ccccgatgtg aagttcatgc ttgcaaacca 480 aatccatgta
aaaacaatgg aagatgttat cccgatggca aaacaggata taaatgtaaa 540
tgtgtcggag gatactcagg acctacatgt caagaaaacg cctgtaaacc aaacccctgt
600 agaaatggtg gaaaatgttc agcagacaaa tttggagact acacatgcga
ttgtcgtcca 660 ggatattttg gtccacaatg cgagaagtat gtgtgtgccc
ctaatccatg taaaaacagc 720 gggatatgtt catctgatgg cagcggtggt
tacagatgta aatgtaaagg aggatactct 780 ggtcctacat gtaaagttaa
tgtctgtaaa cccaacccac gcaagaacag tggcagatgt 840 gtcaacaaag
gcagtagtta caactgtatc tgtaaaggag gttattctgg acctacatgt 900
ggagttaatg cctgtaaacc caatccatgc aagaacagtg gcagatgtgt caacaaaggc
960 agtagttaca actgtatctg taaaggaggt tattctggac ctaaatgtgg
agaacatgtt 1020 tgtaaaccta atccatgcca aaatagaggt cgttgtgttc
ccgaaggacg tgatggttac 1080 agatgtaaat gcgtaggtgg atattccggt
cctacttgtg atgaaaatgt atgtaaaccc 1140 aatccttgtc aaaacaaagg
cagatgctac cctgacaaca gtgatgatgg gtttaaatgt 1200 agatgtttag
gaggatacaa aggtcctaca tgtgaagata aaccaaaccc atgtaacaca 1260
aaaccttgca aaaacggagg aaaatgtaat tataatggaa aaacttatac ctgtaaatgt
1320 gcttacggat atcgtggaag acattgtact gctaaagcat ataaaccaaa
cccatgtgct 1380 tcaagacctt gcaaaaatag aggaaagtgt actgttaaag
gtaccggata tgtgtgtaca 1440 tgtgcaaaag gatatagtgg cagatattgt
gcccttaaat caccaccatc ctacaacgat 1500 gatgacgagt attaagttaa cctagac
1527 2 508 PRT Mytilus edulis 2 Met Leu Phe Ser Phe Phe Leu Leu Leu
Thr Cys Thr Gln Leu Cys Leu 1 5 10 15 Gly Thr Ser Pro Arg Pro Tyr
Asp Asp Asp Glu Asp Asp Tyr Thr Pro 20 25 30 Pro Val Tyr Lys Pro
Ser Pro Ser Arg Tyr Arg Pro Val Asn Pro Cys 35 40 45 Leu Lys Lys
Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Ser Gly Gly 50 55 60 Ser
Tyr Lys Cys Val Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn 65 70
75 80 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg
Cys 85 90 95 Ile Pro Val Gly Lys Thr Phe Lys Cys Glu Cys Arg Asn
Gly Asn Phe 100 105 110 Gly Arg Leu Cys Glu Arg Asn Val Cys Ser Pro
Asn Pro Cys Lys Asn 115 120 125 Lys Gly Lys Cys Ala Pro Leu Gly Lys
Thr Gly Tyr Lys Cys Thr Cys 130 135 140 Ser Gly Gly Tyr Thr Gly Pro
Arg Cys Glu Val His Ala Cys Lys Pro 145 150 155 160 Asn Pro Cys Lys
Asn Asn Gly Arg Cys Tyr Pro Asp Gly Lys Thr Gly 165 170 175 Tyr Lys
Cys Lys Cys Val Gly Gly Tyr Ser Gly Pro Thr Cys Gln Glu 180 185 190
Asn Ala Cys Lys Pro Asn Pro Cys Arg Asn Gly Gly Lys Cys Ser Ala 195
200 205 Asp Lys Phe Gly Asp Tyr Thr Cys Asp Cys Arg Pro Gly Tyr Phe
Gly 210 215 220 Pro Gln Cys Glu Lys Tyr Val Cys Ala Pro Asn Pro Cys
Lys Asn Ser 225 230 235 240 Gly Ile Cys Ser Ser Asp Gly Ser Gly Gly
Tyr Arg Cys Lys Cys Lys 245 250 255 Gly Gly Tyr Ser Gly Pro Thr Cys
Lys Val Asn Val Cys Lys Pro Asn 260 265 270 Pro Arg Lys Asn Ser Gly
Arg Cys Val Asn Lys Gly Ser Ser Tyr Asn 275 280 285 Cys Ile Cys Lys
Gly Gly Tyr Ser Gly Pro Thr Cys Gly Val Asn Ala 290 295 300 Cys Lys
Pro Asn Pro Cys Lys Asn Ser Gly Arg Cys Val Asn Lys Gly 305 310 315
320 Ser Ser Tyr Asn Cys Ile Cys Lys Gly Gly Tyr Ser Gly Pro Lys Cys
325 330 335 Gly Glu His Val Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly
Arg Cys 340 345 350 Val Pro Glu Gly Arg Asp Gly Tyr Arg Cys Lys Cys
Val Gly Gly Tyr 355 360 365 Ser Gly Pro Thr Cys Asp Glu Asn Val Cys
Lys Pro Asn Pro Cys Gln 370 375 380 Asn Lys Gly Arg Cys Tyr Pro Asp
Asn Ser Asp Asp Gly Phe Lys Cys 385 390 395 400 Arg Cys Leu Gly Gly
Tyr Lys Gly Pro Thr Cys Glu Asp Lys Pro Asn 405 410 415 Pro Cys Asn
Thr Lys Pro Cys Lys Asn Gly Gly Lys Cys Asn Tyr Asn 420 425 430 Gly
Lys Thr Tyr Thr Cys Lys Cys Ala Tyr Gly Tyr Arg Gly Arg His 435 440
445 Cys Thr Ala Lys Ala Tyr Lys Pro Asn Pro Cys Ala Ser Arg Pro Cys
450 455 460 Lys Asn Arg Gly Lys Cys Thr Val Lys Gly Thr Gly Tyr Val
Cys Thr 465 470 475 480 Cys Ala Lys Gly Tyr Ser Gly Arg Tyr Cys Ala
Leu Lys Ser Pro Pro 485 490 495 Ser Tyr Asn Asp Asp Asp Glu Tyr Val
Asn Leu Asp 500 505 3 1413 DNA Mytilus edulis 3 atgttgtttt
ctttcttact tttgcttact tgtacccagc tttgcctagg aacgtcacca 60
cgtccatatg atgatgatga ggatgactac tcaccaccag tatacaagcc ttcaccatcg
120 aaatatcgac cagtaaaccc ctgtttaaag aagccatgta aatacaatgg
agtatgtaaa 180 cccaatggtg gctcctataa atgtacctgc aaaggaggat
actatggata caactgcaac 240 ctaaaaaacg catgtaaacc aaaccaatgt
aaaaataaag gtagatgttt acctgttgga 300 aaaacattta aatgtgtatg
tagaaatgga aactttggca gactttgcga aagaaatgta 360 tgtagcccta
atccttgtaa gaacaaagga aagtgcgccc cctggggaaa gacaggatat 420
aaatgtagat gtagtggagg atacactggt ccccgatgtg aagtacatgc ttgcaaacca
480 aacccatgta aaaacaaggg aagttgtaag cccgatggca aaacaggata
taaatgtaca 540 tgtgtcggag gatactcagg acctacatgt caagaaaacg
cttgtaaacc aaacccctgt 600 agcaatggag ggaaatgctc agctgacaaa
tttggagact actcatgcga atgtcaaaaa 660 ggatattatg gtccagaatg
cgagaaatat gtctgtgccc ctaatccatg taaaaacggc 720 gggaaatgtt
cttctgatgg tagcggtggt tacaaatgtc aatgtaccgg aggatactca 780
ggtctaacat gtaatgttaa tgtctgtaaa cccaatccat gcaagaacag tggcagatgt
840 gtcaacaaag gcagtagtta caaatgtatc tgtaaaggag gatattctgg
acctacatgt 900 ggagaacatg tatgtaaacc taatccatgc cagaatagag
gtcgttgtta tcccgaagga 960 cgggatggtt acagatgtaa atgcgtaggt
ggatattccg gtcctacttg tgatgaagat 1020 gtatgtaaac ccaatccatg
tcagaacaaa ggcagatgtt accctgacaa gagtgatgat 1080 gggtttaaat
gtaaatgtct aggaggatac acaggtccta catgtgaaga taaaccaaac 1140
ccatgtaaca caaaaccttg caaaaacgga ggaaaatgta gttataatgg gaaaacttat
1200 acctgtaaat gtgcttatgg ataccgtgga agacattgta ctgctaaagc
atataaccca 1260 tgtgcttcaa gaccttgcaa aaatagagga aagtgtactg
ttaaaggtac caaatatgtg 1320 tgtacatgtg caaaaggata tagtggcaga
tattgtgccc ttaaatcacc accatcctac 1380 aacgatgatg acgagtatta
agttaaccta gac 1413 4 470 PRT Mytilus edulis 4 Met Leu Phe Ser Phe
Leu Leu Leu Leu Thr Cys Thr Gln Leu Cys Leu 1 5 10 15 Gly Thr Ser
Pro Arg Pro Tyr Asp Asp Asp Glu Asp Asp Tyr Ser Pro 20 25 30 Pro
Val Tyr Lys Pro Ser Pro Ser Lys Tyr Arg Pro Val Asn Pro Cys 35 40
45 Leu Lys Lys Pro Cys Lys Tyr Asn Gly Val Cys Lys Pro Asn Gly Gly
50 55 60 Ser Tyr Lys Cys Thr Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn
Cys Asn 65 70 75 80 Leu Lys Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn
Lys Gly Arg Cys 85 90 95 Leu Pro Val Gly Lys Thr Phe Lys Cys Val
Cys Arg Asn Gly Asn Phe 100 105 110 Gly Arg Leu Cys Glu Arg Asn Val
Cys Ser Pro Asn Pro Cys Lys Asn 115 120 125 Lys Gly Lys Cys Ala Pro
Trp Gly Lys Thr Gly Tyr Lys Cys Arg Cys 130 135 140 Ser Gly Gly Tyr
Thr Gly Pro Arg Cys Glu Val His Ala Cys Lys Pro 145 150 155 160 Asn
Pro Cys Lys Asn Lys Gly Ser Cys Lys Pro Asp Gly Lys Thr Gly 165 170
175 Tyr Lys Cys Thr Cys Val Gly Gly Tyr Ser Gly Pro Thr Cys Gln Glu
180 185 190 Asn Ala Cys Lys Pro Asn Pro Cys Ser Asn Gly Gly Lys Cys
Ser Ala 195 200 205 Asp Lys Phe Gly Asp Tyr Ser Cys Glu Cys Gln Lys
Gly Tyr Tyr Gly 210 215 220 Pro Glu Cys Glu Lys Tyr Val Cys Ala Pro
Asn Pro Cys Lys Asn Gly 225 230 235 240 Gly Lys Cys Ser Ser Asp Gly
Ser Gly Gly Tyr Lys Cys Gln Cys Thr 245 250 255 Gly Gly Tyr Ser Gly
Leu Thr Cys Asn Val Asn Val Cys Lys Pro Asn 260 265 270 Pro Cys Lys
Asn Ser Gly Arg Cys Val Asn Lys Gly Ser Ser Tyr Lys 275 280 285 Cys
Ile Cys Lys Gly Gly Tyr Ser Gly Pro Thr Cys Gly Glu His Val 290 295
300 Cys Lys Pro Asn Pro Cys Gln Asn Arg Gly Arg Cys Tyr Pro Glu Gly
305 310 315 320 Arg Asp Gly Tyr Arg Cys Lys Cys Val Gly Gly Tyr Ser
Gly Pro Thr 325 330 335 Cys Asp Glu Asp Val Cys Lys Pro Asn Pro Cys
Gln Asn Lys Gly Arg 340 345 350 Cys Tyr Pro Asp Lys Ser Asp Asp Gly
Phe Lys Cys Lys Cys Leu Gly 355 360 365 Gly Tyr Thr Gly Pro Thr Cys
Glu Asp Lys Pro Asn Pro Cys Asn Thr 370 375 380 Lys Pro Cys Lys Asn
Gly Gly Lys Cys Ser Tyr Asn Gly Lys Thr Tyr 385 390 395 400 Thr Cys
Lys Cys Ala Tyr Gly Tyr Arg Gly Arg His Cys Thr Ala Lys 405 410 415
Ala Tyr Asn Pro Cys Ala Ser Arg Pro Cys Lys Asn Arg Gly Lys Cys 420
425 430 Thr Val Lys Gly Thr Lys Tyr Val Cys Thr Cys Ala Lys Gly Tyr
Ser 435 440 445 Gly Arg Tyr Cys Ala Leu Lys Ser Pro Pro Ser Tyr Asn
Asp Asp Asp 450 455 460 Glu Tyr Val Asn Leu Asp 465 470 5 1362 DNA
Mytilus edulis 5 atgttgtttt ctttctttct tttgcttact tgtacccagc
tttgcctagg aacgtcacca 60 cgtcaatatg atgatgatga ggatgactac
tcaccaccag tatacaagcc ttcaccatcg 120 aaatatcgac cagtaaaccc
ctgtttaaag aagccatgca aatacaatgg agtatgtaaa 180 cccaatggtg
gctcctataa atgtacctgc aaaggaggat actatggata caactgcaac 240
ctaaaaaacg catgtaaacc aaaccaatgt aaaaataaaa gtagatgtat acctgttgga
300 aaaacatata aatgtgtatg tagaaatgga aactatggca gactttgcga
aaaaaatgta 360 tgtagcccta atccttgtaa gaacaaagga aagtgcgccc
cctggggaaa gacaggatat 420 aaatgtagat gtagtggagg atacactggt
ccccgatgtg aagtacatgc ttgcaaacca 480 aacccatgta aaaacaatgg
aagttgtaag cccgatggca aaacaggata taaatgtaca 540 tgtgtcggag
gatactcagg acctacatgt caagaaaacg cctgtaaacc aaacccctgt 600
agcaatggag ggaaatgctc agctgacaaa tttggagact actcatgcga atgtcaaaaa
660 agatattatg gtccagaatg cgagaaatat gtctgtgccc ctaatccatg
taaaaatggc 720 gggaaatgtt cttctgatgg tagcggtggt tacaaatgtc
aatgtaccgg aggatactca 780 ggtctaacat gtaatgttaa tgtctgtaaa
cccaatccat gcaagaacag tggcagatgt 840 gtcaacaaag gcagtagtta
caaatgtatc tgtaaaggag gatattctgg acctacatgt 900 ggagaaaatg
tatgtaaacc caatccatgt cagaacaaag gcagatgtta ccctgaccag 960
agtgatgatg ggtttaaatg taaatgtcta ggaggataca caggtcctac atgtgaagat
1020 aaaccaaacc catgtaacac aaaaccttgc aaaaacggag gaaaatgtag
ttataatggg 1080 aaaacttata cctgtaaatg tgcttatgga taccgtggaa
gacattgtac tgctaaagca 1140 tataacccat gtgcttcaag accttgcaaa
aatagaggaa agtgtactgt taaaggtacc 1200 aaatatgtgt gtacatgtgc
aaaaggatat agtggcagat attgtgccct taaatcacca 1260 ccatcctaca
acgatgatga cgagtattaa gttaacctag aaatttaaaa ttgtttttca 1320
ttattaaagg atatttgata ttcaaaaaaa aaaaaaaaaa aa 1362 6 451 PRT
Mytilus edulis 6 Met Leu Phe Ser Phe Phe Leu Leu Leu Thr Cys Thr
Gln Leu Cys Leu 1 5 10 15 Gly Thr Ser Pro Arg Gln Tyr Asp Asp Asp
Glu Asp Asp Tyr Ser Pro 20 25 30 Pro Val Tyr Lys Pro Ser Pro Ser
Lys Tyr Arg Pro Val Asn Pro Cys 35 40 45 Leu Lys Lys Pro Cys Lys
Tyr Asn Gly Val Cys Lys Pro Asn Gly Gly 50 55 60 Ser Tyr Lys Cys
Thr Cys Lys Gly Gly Tyr Tyr Gly Tyr Asn Cys Asn 65 70 75 80 Leu Lys
Asn Ala Cys Lys Pro Asn Gln Cys Lys Asn Lys Ser Arg Cys 85 90 95
Ile Pro Val Gly Lys Thr Tyr Lys Cys Val Cys Arg Asn Gly Asn Tyr 100
105 110 Gly Arg Leu Cys Glu Lys Asn Val Cys Ser Pro Asn Pro Cys Lys
Asn 115 120 125 Lys Gly Lys Cys Ala Pro Trp Gly Lys Thr Gly Tyr Lys
Cys Arg Cys 130 135 140 Ser Gly Gly Tyr Thr Gly Pro Arg Cys Glu Val
His Ala Cys Lys Pro 145 150 155 160 Asn Pro Cys Lys Asn Asn Gly Ser
Cys Lys Pro Asp Gly Lys Thr Gly 165 170 175 Tyr Lys Cys Thr Cys Val
Gly Gly Tyr Ser Gly Pro Thr Cys Gln Glu 180 185 190 Asn Ala Cys Lys
Pro Asn Pro Cys Ser Asn Gly Gly Lys Cys Ser Ala 195 200 205 Asp Lys
Phe Gly Asp Tyr Ser Cys Glu Cys Gln Lys Arg Tyr Tyr Gly 210 215 220
Pro Glu Cys Glu Lys Tyr Val Cys Ala Pro Asn Pro Cys Lys Asn Gly 225
230 235 240 Gly Lys Cys Ser Ser Asp Gly Ser Gly Gly Tyr Lys Cys Gln
Cys Thr 245 250 255 Gly Gly Tyr Ser Gly Leu Thr Cys Asn Val Asn Val
Cys Lys Pro Asn 260 265 270 Pro Cys Lys Asn Ser Gly Arg Cys Val Asn
Lys Gly Ser Ser Tyr Lys 275 280 285 Cys Ile Cys Lys Gly Gly Tyr Ser
Gly Pro Thr Cys Gly Glu Asn Val 290 295 300 Cys Lys Pro Asn Pro Cys
Gln Asn Lys Gly Arg Cys Tyr Pro Asp Gln 305 310 315 320 Ser Asp Asp
Gly Phe Lys Cys Lys Cys Leu Gly Gly Tyr Thr Gly Pro 325 330 335 Thr
Cys Glu Asp Lys Pro Asn Pro Cys Asn Thr Lys Pro Cys Lys Asn 340 345
350 Gly Gly Lys Cys Ser Tyr Asn Gly Lys Thr Tyr Thr Cys Lys Cys Ala
355 360 365 Tyr Gly Tyr Arg Gly Arg His Cys Thr Ala Lys Ala Tyr Asn
Pro Cys 370 375 380 Ala Ser Arg Pro Cys Lys Asn Arg Gly Lys Cys Thr
Val Lys Gly Thr 385 390 395 400 Lys Tyr Val Cys Thr Cys Ala Lys Gly
Tyr Ser Gly Arg Tyr Cys Ala 405 410 415 Leu Lys Ser Pro Pro Ser Tyr
Asn Asp Asp Asp Glu Tyr Val Asn Leu 420 425 430 Glu Ile Asn Cys Phe
Ser Leu Leu Lys Asp Ile Tyr Ser Lys Lys Lys 435 440 445 Lys Lys Lys
450 7 20 DNA Artificial T7 Sequencing Primer 7 taatacgact
cactataggg 20 8 21 DNA Artificial V5C-term Reverse Sequencing
Primer 8 accgaggaga gggttaggga t 21 9 18 DNA Artificial Sequencing
Primer 506-(HS-PYes2) 9 tttcggttag agcggatg 18 10 20 DNA Artificial
Sequencing Primer 507 (HS-PYes-2) 10 agggcgtgaa tgtaagcgtg 20 11 18
DNA Artificial Sequencing Primer 508 (HS-Mefp-2 internal) 11
tttggtccag aatgcgag 18 12 17 DNA Artificial Sequencing Primer Sq1
(HS-Mefp-2 internal) 12 ctttggcaga ctttgcg 17 13 17 DNA Artificial
Sequencing Primer SQ-2 (HS-Mefp-2 interna) 13 acggaaagtg ctcaccc 17
14 17 DNA Artificial Sequencing Primer SQ-3 (HS-Mefp-2 internal) 14
aagtgctcac ccttggg 17 15 21 DNA Artificial Sequencing Primer FP-1
(Epicentre) 15 cccaatacgc aaaccgcctc t 21 16 25 DNA Artificial
Sequencing Primer RP-1 (Epicentre) 16 ttagaaaaat aaacaaatag gggtt
25 17 31 DNA Artificial PCR Primer 514-Mefp-2 17 gcggccgcca
cagaagcatc atgttgtttt c 31 18 30 DNA Artificial PCR Primer
515-Mefp-2 18 gagctcgtct aggttaactt aatactcgtc 30 19 1489 DNA
Mytilus galloprovincialis 19 gtcacagaag catcatgttg ttttctttct
ttcttttgct tacttgtacc cagctttgcc 60 taggaactaa tcgacctgat
tataatgatg atgaggagga tgactacaaa ccaccagtat 120 acaagccttc
accatcgaaa tatcgaccag taaacccctg tttaaagaag ccatgtaaat 180
acaatggagt atgtaaaccc agaggtggct cctacaaatg tttctgcaaa ggaggatact
240 atggatacaa ttgcaaccta aaaaacgcat gtaaaccaaa ccaatgtaaa
aataaaagta 300 gatgtgtacc tgttggaaaa acatttaaat gtgtatgtag
aaatggaaac tttggcagac 360 tttgcgaaaa aaatgtatgt agcccgaatc
cttgtaaaaa
caacggaaag tgctcaccct 420 tgggaaagac aggatataaa tgtacatgta
gtggaggata cactggtccc cgatgtgaag 480 tgcatgcttg caaaccaaat
ccatgtaaaa acaaaggaag atgttttccc gatggtaaaa 540 cagggtataa
atgtagatgt gtcgacggat actcaggacc tacatgtcag gaaaacgcct 600
gtaaaccaaa cccatgtagc aatggaggga catgctcagc tgacaaattt ggagactact
660 catgcgaatg tcgcccagga tattttggtc cagaatgcga gaggtatgtg
tgtgccccta 720 atccatgtaa aaacggcggc atatgttcat ctgatggcag
cggcggttac agatgtagat 780 gtaaaggagg atactctggt cctacatgta
aagtaaatgt ctgtaaaccc actccatgca 840 agaacagtgg cagatgtgtc
aacaaaggca gtagttacaa ctgtatctgt aaaggaggtt 900 attctgggcc
tacatgtgga gaaaatgtat gtaaacccaa tccatgtcaa aacagaggca 960
gatgttaccc tgacaacagt gatgatgggt ttaaatgtag atgtgtagga ggttacaaag
1020 gtcctacatg tgaagataaa ccaaacccat gcaacacaaa accttgcaaa
aatggaggaa 1080 aatgtaatta taatggaaaa atttatacct gtaaatgtgc
atacggatgg cgtggacgac 1140 attgtactga taaagcttat aaaccaaacc
cttgtgttgt ttcaaagcca tgcaaaaaca 1200 gaggaaagtg tatttggaat
ggaaaagctt atagatgcaa atgcgcatat ggatacggcg 1260 gcaggcattg
cactaaaaaa tcatataaaa aaaacccatg tgcttcacgt ccatgtaaaa 1320
atagaggaaa gtgtacagat aaaggaaacg gatatgtgtg taaatgtgcc agaggataca
1380 gtggcagata ttgttctcta aaatcaccac catcctacga cgatgacgag
tattaagtta 1440 acctagacat ttaaaattgt ttttcattat taaaggatat
ttgatattc 1489 20 4 PRT Artificial Amino Acid Sequence ofor 514
primer 20 Met Leu Phe Ser 1 21 4 PRT Artificial Amino Acid Sequence
for Primer 515 21 Asp Glu Tyr Xaa 1 22 6 PRT Mytilus edulis 22 Ala
Lys Pro Thr Tyr Lys 1 5 23 10 PRT Mytilus edulis MOD_RES (3)..(3)
where P in residue 3 is trans-4-hydroxy-L- proline 23 Ala Lys Pro
Ser Tyr Pro Pro Thr Tyr Lys 1 5 10
* * * * *