U.S. patent application number 10/788346 was filed with the patent office on 2005-10-13 for human neuronal attachment factor-1.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Dillon, Patrick J., Hastings, Gregg.
Application Number | 20050227237 10/788346 |
Document ID | / |
Family ID | 26682485 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050227237 |
Kind Code |
A9 |
Hastings, Gregg ; et
al. |
October 13, 2005 |
Human neuronal attachment factor-1
Abstract
A human F-spondin-like protein and DNA (RNA) encoding such
protein and a procedure for producing such protein by recombinant
techniques is disclosed. Also disclosed are methods for utilizing
such polypeptide for treating spinal cord injuries and damage to
peripheral nerves by promoting neural-cell adhesion and neurite
extension, inhibiting tumor metastases and tumor angiogenesis, and
stimulating wound repair. Antagonists are also disclosed which may
be utilized to prevent malaria. Diagnostic assays for identifying
mutations in nucleic acid sequence encoding a polypeptide of the
present invention and for detecting altered levels of the
polypeptide of the present invention for detecting diseases, for
example, cancer, are also disclosed.
Inventors: |
Hastings, Gregg; (Westlake
Village, CA) ; Dillon, Patrick J.; (Carlsbad,
CA) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
INTELLECTUAL PROPERTY DEPT.
14200 SHADY GROVE ROAD
ROCKVILLE
MD
20850
US
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
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Prior
Publication: |
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Document Identifier |
Publication Date |
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US 0146928 A1 |
July 29, 2004 |
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Family ID: |
26682485 |
Appl. No.: |
10/788346 |
Filed: |
March 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10788346 |
Mar 1, 2004 |
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09170042 |
Oct 13, 1998 |
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6759512 |
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09170042 |
Oct 13, 1998 |
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08799173 |
Feb 12, 1997 |
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5871969 |
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60011519 |
Feb 12, 1996 |
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Current U.S.
Class: |
435/6.16 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101;
Y02A 50/411 20180101; Y02A 50/30 20180101; A61K 38/00 20130101 |
Class at
Publication: |
435/006 ;
530/350; 435/320.1; 435/325; 536/023.5; 435/069.1 |
International
Class: |
C07K 014/47; C12Q
001/68; C07H 021/04 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule nucleic acid molecule
comprising a polynucleotide having a nucleotide sequence at least
95% identical to a sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a full-length NAF-1 polypeptide
having the complete amino acid sequence in SEQ ID NO:2, or the
complete amino acid sequence encoded by the cDNA clone contained in
the ATCC Deposit No. 97343; (b) a nucleotide sequence encoding a
full-length NAF-1 polypeptide having the complete amino acid
sequence in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,
positions 1 to 331 of SEQ ID NO:2) or the complete amino acid
sequence excepting the N-terminal methionine encoded by the cDNA
clone contained in the ATCC Deposit No. 97343; (c) a nucleotide
sequence encoding a predicted mature form of the NAF-1 polypeptide
having the amino acid sequence at positions 24-331 or 27-331 in SEQ
ID NO:2 or as encoded by the cDNA clone contained in the ATCC
Deposit No. 97343; (d) a nucleotide sequence encoding a polypeptide
comprising the predicted TSR domain of the NAF-1 polypeptide having
the amino acid sequence at positions 284-330 in SEQ ID NO:2 or as
encoded by the cDNA clone contained in the ATCC Deposit No. 97343;
and (e) a nucleotide sequence complementary to any of the
nucleotide sequences in (a), (b), (c) or (d) above.
2. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the complete nucleotide sequence in FIG. 1 (SEQ ID NO:1).
3. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence in FIG. 1 (SEQ ID NO:1) encoding the
NAF-1 polypeptide having the amino acid sequence in positions 2 to
331 of SEQ ID NO:2.
4. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence in FIG. 1 (SEQ ID NO: 1) encoding the
mature NAF-1 polypeptide having the amino acid sequence from about
27 to about 331 in SEQ ID NO:2.
5. An isolated nucleic acid molecule comprising a polynucleotide
having a nucleotide sequence at least 95% identical to a sequence
selected from the group consisting of: (a) a nucleotide sequence
encoding a polypeptide comprising the amino acid sequence of
residues n-331 of SEQ ID NO:2, where n is an integer in the range
of 1-283; (b) a nucleotide sequence encoding a polypeptide
comprising the amino acid sequence of residues 1-m of SEQ ID NO:2,
where m is either 330 or 331; (c) a nucleotide sequence encoding a
polypeptide having the amino acid sequence consisting of residues
n-m of SEQ ID NO:2, where n and m are integers as defined
respectively in (a) and (b) above; and (d) a nucleotide sequence
encoding a polypeptide consisting of a portion of the complete
NAF-1 amino acid sequence encoded by the cDNA clone contained in
ATCC Deposit No. 97343 wherein said portion excludes from 1 to
about 283 amino acids from the amino terminus of said complete
amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. 97343; (e) a nucleotide sequence encoding a polypeptide
consisting of a portion of the complete NAF-1 amino acid sequence
encoded by the cDNA clone contained in ATCC Deposit No. 97343
wherein said portion excludes 1 amino acid from the carboxy
terminus of said complete amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 97343; and (f) a nucleotide
sequence encoding a polypeptide consisting of a portion of the
complete NAF-1 amino acid sequence encoded by the cDNA clone
contained in ATCC Deposit No. 97343 wherein said portion include a
combination of any of the amino terminal and carboxy terminal
deletions in (d) and (e), above.
6. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the complete nucleotide sequence of the cDNA clone contained in
ATCC Deposit No. 97343.
7. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding the NAF-1 polypeptide having
the complete amino acid sequence excepting the N-terminal
methionine encoded by the cDNA clone contained in ATCC Deposit No.
97343.
8. The nucleic acid molecule of claim 1 wherein said polynucleotide
has the nucleotide sequence encoding the mature polypeptide having
the amino acid sequence encoded by the cDNA clone contained in ATCC
Deposit No. 97343.
9. An isolated nucleic acid molecule comprising a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide having a nucleotide sequence identical to a
nucleotide sequence in (a), (b), (c), (d) or (e) of claim 1 wherein
said polynucleotide which hybridizes does not hybridize under
stringent hybridization conditions to a polynucleotide having a
nucleotide sequence consisting of only A residues or of only T
residues.
10. An isolated nucleic acid molecule comprising a polynucleotide
which encodes the amino acid sequence of an epitope-bearing portion
of a NAF-1 polypeptide having an amino acid sequence in (a), (b),
(c) or (d) of claim 1.
11. The isolated nucleic acid molecule of claim 10, which encodes
an epitope-bearing portion of a NAF-1 polypeptide wherein the amino
acid sequence of said portion is selected from the group of
sequences in SEQ ID NO:2 consisting of: a polypeptide comprising
amino acid residues from about Pro-75 to about Gly-100; a
polypeptide comprising amino acid residues from about Thr-168 to
about Leu-180; a polypeptide comprising amino acid residues from
about Asp-204 to about Ile-226; a polypeptide comprising amino acid
residues from about Ile-258 to about Pro-281; and a polypeptide
comprising amino acid residues from about Glu-291 to about
Ser-327.
12. A method for making a recombinant vector comprising inserting
an isolated nucleic acid molecule of claim 1 into a vector.
13. A recombinant vector produced by the method of claim 12.
14. A method of making a recombinant host cell comprising
introducing the recombinant vector of claim 13 into a host
cell.
15. A recombinant host cell produced by the method of claim 14.
16. A recombinant method for producing a NAF-1 polypeptide,
comprising culturing the recombinant host cell of claim 15 under
conditions such that said polypeptide is expressed and recovering
said polypeptide.
17. An isolated NAF-1 polypeptide comprising an amino acid sequence
at least 95% identical to a sequence selected from the group
consisting of: (a) the amino acid sequence of the full-length NAF-1
polypeptide having the complete amino acid sequence shown in SEQ ID
NO:2 or the complete amino acid sequence excepting the N-terminal
methionine encoded by the cDNA clone contained in the ATCC Deposit
No. 97343; (b) the amino acid sequence of the full-length NAF-1
polypeptide having the complete amino acid sequence shown in SEQ ID
NO:2 excepting the N-terminal methionine (i.e., positions 1-331 of
SEQ ID NO:2) or the complete amino acid sequence excepting the
N-terminal methionine encoded by the cDNA clone contained in the
ATCC Deposit No. 97343; (c) the amino acid sequence of the mature
NAF-1 polypeptide having the amino acid sequence of residues 24-331
or 27-331 in SEQ ID NO:2, or the mature NAF-1 amino acid sequence
as encoded by the cDNA clone contained in ATCC Deposit No. 97343;
and (d) the amino acid sequence of the TSR domain of NAF-1 having
the amino acid sequence of residues 284 to 330 of SEQ ID NO:2, or
the amino acid sequence of the TSR domain of NAF-1 encoded by the
cDNA clone contained in ATCC Deposit No. 97343.
18. An isolated polypeptide comprising an epitope-bearing portion
of the NAF-1 protein, wherein said portion is selected from the
group consisting of: a polypeptide comprising amino acid residues
from about Pro-75 to about Gly-100; a polypeptide comprising amino
acid residues from about Thr-168 to about Leu-180; a polypeptide
comprising amino acid residues from about Asp-204 to about Ile-226;
a polypeptide comprising amino acid residues from about Ile-258 to
about Pro-281; and a polypeptide comprising amino acid residues
from about Glu-291 to about Ser-327.
19. An isolated antibody that binds specifically to a NAF-1
polypeptide of claim 17.
20. An isolated nucleic acid molecule comprising a polynucleotide
having a sequence at least 95% identical to a sequence selected
from the group consisting of: (a) the nucleotide sequence of clone
HLHCE24R (shown as SEQ ID NO: 15); (b) the nucleotide sequence of
clone HLHDR83R (shown as SEQ ID NO: 16); (c) the nucleotide
sequence of clone HPTSB36R (shown as SEQ ID NO: 17); (d) the
nucleotide sequence of a portion of the sequence shown in FIG. 1
(SEQ ID NO: 1) wherein said portion comprises at least 50
contiguous nucleotides from nucleotide 1 to 650; and (e) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c) and (d).
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
09/170,042, filed Oct. 13, 1998, which is a division of U.S.
application Ser. No. 08/799,173, filed Feb. 12, 1997 (now U.S. Pat.
No. 5,871,969, issued Feb. 16, 1999), which claims benefit under 35
U.S.C. .sctn. 119(e) of U.S. Provisional Application No.
60/011,519, filed on Feb. 12, 1996.
FIELD OF THE INVENTION
[0002] This invention relates to newly identified polynucleotides,
polypeptides encoded by such polynucleotides, the use of such
polynucleotides and polypeptides, as well as the production of such
polynucleotides and polypeptides. More particularly, the
polypeptide of the present invention has been putatively identified
as a human neuronal attachment factor-1, sometimes hereinafter
referred to as "NAF-1". The invention also relates to inhibiting
the action of such polypeptides.
BACKGROUND OF THE INVENTION
[0003] F-spondin (FSP) is a gene that is predominantly expressed
during the early development of the vertebrate nervous system. The
main function is thought to be in neural cell pattern formation and
axonal growth. It was found in a subtractive hybridization screen
designed to isolate floor-plate specific genes. The floor-plate
provides diffusible signals that act on the neurons that extend
from the developing spinal cord. These signals can lead to
chemoattraction and fasciculation of commissural axons in the
ventral midline. F-spondin mRNA is expressed at high levels in the
developing neural tube at the ventral midline even before cell
differentiation markers can detect the floor-plate. F-spondin is
not detectable in other regions of the spinal cord until later in
embryonic life. There is also transient F-spondin expression early
in peripheral nerve development which diminishes to undetectable
levels following birth. The adult central nervous system contains
F-spondin while the peripheral nerve (sciatic nerve) does not.
Outside the adult nervous system, organs such as the lung and
kidney also express F-spondin. The protein is 807 amino acids and
codes for a predicted 90 kD polypeptide. The apparent size is
approximately 116 kD by SDS-PAGE which indicates post-translational
modifications such as glycosylation. There are six domains
homologous to the thrombospondin (TSP) type 1 repeats (TSR) which
have been shown to control cell adhesion. The protein has been
expressed in COS cells and purified as a myc-tag fusion protein.
This protein was active in promoting neurite extension and adhesion
of embryonic dorsal root ganglion and dorsal spinal cords
respectively. It was not chemotropic for embryonic dorsal spinal
cord neurons. (Klar, A. et al., Cell, 69:95-110 (1992)).
[0004] The C-terminal half of F-spondin contains 6 repeats
identified in thrombospondin and other proteins implicated in cell
adhesion. Thrombospondin is a 450,000-dalton glyco-protein secreted
by platelets in response to such physiological activators as
thrombin and collagen (Lawler, J., Blood, 67:1197-1209 (1986)). TSP
comprises 3% of the total platelet protein and 25% of the total
platelet-secreted proteins (Tuszynski, G. P., et al., J. Biol.
Chem., 260:12240-12245 (1985)). Although the precise biological
role of TSP has yet to be fully established, it is generally
accepted that TSP plays a major role in cell adhesion and cell-cell
interactions. It should be pointed out that the C-terminal repeats
present in thrombospondin may have different biological
activities.
[0005] TSP was found to promote the cell-substratum adhesion of a
variety of cells, including platelets, melanoma cells, smooth
muscle cells, endothelial cells, fibroblasts and epithelial cells
(Tuszynski, G. P., et al., Science (Washington, D.C.),
236:1570-1573 (1983)).
[0006] Thrombospondin has been postulated to play a role in
malarial infection induced by only one strain of malaria,
plasmodium falciparum. During malarial infection, TSP promotes
adhesion of parasitized red cells to endothelial cells (Roberts, D.
D., et al., Nature (Lond.), 318:64-66 (1984)) and during tumor cell
metastases TSP promotes adhesion of mouse sarcoma cells to the
vascular bed and expression of the malignant phenotype of small
cell carcinoma (Castle, V. J., J. Clin. Invest., 87:1883-1883
(1991)).
[0007] Properdin is a complement-binding protein which also
contains the 6 terminal repeats found in thrombospondin. UNC-5, a
C. elegans gene that bears two terminal repeats, appears to guide
the axonal extension of the sub-set of neurons. These proteins,
which contain at least one member of the six terminal repeats, form
a family of proteins which have related functions.
[0008] The gene and polypeptide encoded thereby of the present
invention has been putatively identified as an Neuronal Attachment
Factor-i protein as a result of amino acid sequence homology to rat
F-spondin.
SUMMARY OF THE INVENTION
[0009] In accordance with one aspect of the present invention,
there is provided a novel mature polypeptide, as well as
biologically active and diagnostically or therapeutically useful
fragments, analogs and derivatives thereof. The polypeptide of the
present invention is of human origin.
[0010] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules encoding a
polypeptide of the present invention including mRNAs, cDNAs,
genomic DNAs as well as analogs and biologically active and
diagnostically or therapeutically useful fragments thereof.
[0011] In accordance with another aspect of the present invention
there is provided an isolated nucleic acid molecule encoding a
mature polypeptide expressed by the human cDNA contained in ATCC
Deposit No. 97343.
[0012] In accordance with yet a further aspect of the present
invention, there is provided a process for producing such
polypeptide by recombinant techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells, containing a
nucleic acid sequence encoding a polypeptide of the present
invention, under conditions promoting expression of said protein
and subsequent recovery of said protein.
[0013] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such
polypeptide, or polynucleotide encoding such polypeptide for
therapeutic purposes, for example, to treat spinal cord injuries or
damage to peripheral nerves by promoting neural cell adhesion and
neurite extension, to inhibit tumor cell metastases, inhibit
endothelial cell proliferation, adhesion and motility, to decrease
tumor neovascularization, to be angiostatic for tumor cells and to
promote wound healing.
[0014] In accordance with yet a further aspect of the present
invention, there are provided antibodies against such polypeptides,
which would bind to and neutralize NAF-1 to inhibit its putative
cell adhesion properties to restrict metastases, particularly tumor
metastases.
[0015] In accordance with another aspect of the present invention,
there are provided NAF-1 agonists which mimic NAF-1 and binds to
the NAF-1 receptors.
[0016] In accordance with yet another aspect of the present
invention, there are provided antagonists to such polypeptides,
which may be used to inhibit the action of such polypeptides, for
example, in the treatment of malarial infection caused by
Plasmodium falciparum.
[0017] In accordance with yet a further aspect of the present
invention, there is also provided nucleic acid probes comprising
nucleic acid molecules of sufficient length to hybridize to a
nucleic acid sequence of the present invention.
[0018] In accordance with still another aspect of the present
invention, there are provided diagnostic assays for detecting
diseases or susceptibility to diseases related to mutations in the
nucleic acid sequences encoding a polypeptide of the present
invention.
[0019] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such
polypeptides, or polynucleotides encoding such polypeptides, for in
vitro purposes related to scientific research, for example,
synthesis of DNA and manufacture of DNA vectors.
[0020] These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
[0022] FIG. 1 is an illustration of the cDNA (SEQ ID NO:1) and
corresponding amino acid sequence (SEQ ID NO:2) of the polypeptide
of the present invention. Sequencing was performed using a 373
automated DNA sequencer (Applied Biosystems, Inc.). The putative
leader sequence region is underlined.
[0023] FIG. 2 is an amino acid sequence comparison between the
polypeptide of the present invention (bottom line) (SEQ ID NO:2)
and rat F-spondin (rFSP) (top line) (SEQ ID NO:7).
[0024] FIG. 3 is an amino acid sequence comparison between the cell
adhesion sequence of NAF-1 (FLP-TSR; SEQ ID NO: 18) and the six
cell adhesion sequences of rat F-spondin (FSR-TSR-1, -2, -3, -4,
-5, and -6; SEQ ID NOS:8-13, respectively). Also shown is a TSR
consensus sequence shown in the sequence listing as SEQ ID NO:
14.
[0025] FIG. 4 shows an analysis of the NAF-1 amino acid sequence
(SEQ ID NO:2). Alpha, beta, turn and coil regions; hydrophilicity
and hydrophobicity; amphipathic regions; flexible regions;
antigenic index and surface probability are shown. In the
"Antigenic Index--Jameson-Wolf" graph, the positive peaks indicate
locations of the highly antigenic regions of the NAF-1 protein,
i.e., regions from which epitope-bearing peptides of the invention
can be obtained.
DETAILED DESCRIPTION
[0026] In accordance with an aspect of the present invention, there
is provided an isolated nucleic acid (polynucleotide) which encodes
for the mature polypeptide having the deduced amino acid sequence
of FIG. 1 (SEQ ID NO:2).
[0027] The polynucleotide of this invention was discovered in a
cDNA library derived from human epithelioid sarcoma. It is
structurally related to the rat F-spondin family. It contains an
open reading frame encoding a protein of 331 amino acid residues.
The protein exhibits the highest degree of homology to rat
F-spondin with 33.1% identity and 52.9% similarity over the entire
amino acid stretch. The gene of the present invention shows the
greatest homology at the nucleotide level to the rat F-spondin gene
with 66% similarity and 66% identity. It is also important that the
polypeptide of the present invention contains the conserved motif,
WSXW, which is a potential binding sequence for polypeptides in
this family.
[0028] Northern blot analysis of the protein of the present
invention showed a broad band at 1.6-1.9 kb in liver and lower
level expression in kidney, lung, heart and placenta. Brain
expression was barely detectable. Two libraries which were
constructed from tissues induced to undergo apoptosis, apoptotic
t-cells (HTG) and TNF induced amniotic cells (HAU), had one clone
in each. By extrapolation, NAF-1 was represented at least 50 times
more frequently in apoptotic t-cells expressed sequence tags than
all normal and activated t-cell libraries. In the TNF induced
amniotic cells library, NAF-1 was detected 1 out of 2,414 expressed
sequence tags versus 0 out of 3,595 expressed sequence tags for the
non-TNF treated amniotic cell library.
[0029] The NAF-1 cDNA contains an open reading frame encoding a
polypeptide of 35.8 kD. Amino acids 1-23 and 1-26 encode putative
signal peptides. Accordingly, there are two species of predicted
mature NAF-1 polypeptides one having 311 and the other 314 amino
acids. NAF-1 also contains a putative N-linked glycosylation site
at position 303. The homology of NAF-1 to FSP covers amino acids
199-495 of the latter protein. Thus, NAF-1 does not appear to be
the human counterpart of the rat FSP. NAF-1 contains only one TSR
which begins at amino acid 278. This region is much more homologous
to FSP type 1 repeats than to those of TSP, 38% versus 20%,
respectively. The homology between the NAF-1 TSR and the six FSP
type-1 repeats is shown in FIG. 3. The amino terminal 277 amino
acids of NAF-1 share homology to FSP but show no resemblance to any
other known proteins.
[0030] In accordance with another aspect of the present invention
there are provided isolated polynucleotides encoding a mature
polypeptide expressed by the human cDNA contained in ATCC Deposit
No. 97343, deposited with the American Type Culture Collection
(ATCC), Patent Depository, 10801 University Boulevard, Manassas,
Va., USA (present address), on Nov. 20, 1995. The deposited
material is a pBluescript SK (-) (Stratagene, La Jolla, Calif.)
plasmid that contains the full-length NAF-1 cDNA. The NAF-1 cDNA
has been cloned into the EcoRI, XhoI site.
[0031] The deposit has been made under the terms of the Budapest
Treaty on the International Recognition of the Deposit of
Micro-organisms for purposes of Patent Procedure. The strain will
be irrevocably and without restriction or condition released to the
public upon the issuance of a patent. The deposit is provided
merely as convenience to those of skill in the art and are not an
admission that a deposit is required under 35 U.S.C. .sctn.112. The
sequence of the polynucleotide contained in the deposited material,
as well as the amino acid sequence of the polypeptides encoded
thereby, are controlling in the event of any conflict with any
description of sequences herein. A license may be required to make,
use or sell the deposited material, and no such license is hereby
granted.
[0032] The polynucleotide of the present invention may be in the
form of RNA or in the form of DNA, which DNA includes cDNA, genomic
DNA, and synthetic DNA. The DNA may be double-stranded or
single-stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand. The coding sequence which encodes
the mature polypeptide may be identical to the coding sequence
shown in FIG. 1 (SEQ ID NO:1) or may be a different coding sequence
which coding sequence, as a result of the redundancy or degeneracy
of the genetic code, encodes the same mature polypeptide as the DNA
of FIG. 1 (SEQ ID NO: 1).
[0033] The polynucleotide which encodes for the mature polypeptide
of FIG. 1 (SEQ ID NO:2) may include, but is not limited to: only
the coding sequence for the mature polypeptide; the coding sequence
for the mature polypeptide and additional coding sequence such as a
leader or secretory sequence or a proprotein sequence; the coding
sequence for the mature polypeptide (and optionally additional
coding sequence) and non-coding sequence, such as introns or
non-coding sequence 5' and/or 3' of the coding sequence for the
mature polypeptide.
[0034] Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequence
for the polypeptide as well as a polynucleotide which includes
additional coding and/or non-coding sequence.
[0035] The present invention further relates to variants of the
hereinabove described polynucleotides which encode for fragments,
analogs and derivatives of the polypeptide having the deduced amino
acid sequence of FIG. 1 (SEQ ID NO:2). The variant of the
polynucleotide may be a naturally occurring allelic variant of the
polynucleotide or a non-naturally occurring variant of the
polynucleotide.
[0036] Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as shown in FIG. 1 (SEQ ID
NO:2) as well as variants of such polynucleotides which variants
encode for a fragment, derivative or analog of the polypeptide of
FIG. 1 (SEQ ID NO:2). Such nucleotide variants include deletion
variants, substitution variants and addition or insertion
variants.
[0037] As hereinabove indicated, the polynucleotide may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequence shown in FIG. 1 (SEQ ID NO: 1). As known in the
art, an allelic variant is an alternate form of a polynucleotide
sequence which may have a substitution, deletion or addition of one
or more nucleotides, which does not substantially alter the
function of the encoded polypeptide.
[0038] The present invention also includes polynucleotides, wherein
the coding sequence for the mature polypeptide may be fused in the
same reading frame to a polynucleotide sequence which aids in
expression and secretion of a polypeptide from a host cell, for
example, a leader sequence which functions as a secretory sequence
for controlling transport of a polypeptide from the cell. The
polypeptide having a leader sequence is a preprotein and may have
the leader sequence cleaved by the host cell to form the mature
form of the polypeptide. The polynucleotides may also encode for a
proprotein which is the mature protein plus additional 5' amino
acid residues. A mature protein having a prosequence is a
proprotein and is an inactive form of the protein. Once the
prosequence is cleaved an active mature protein remains. Thus, for
example, the polynucleotide of the present invention may encode for
a mature protein, or for a protein having a prosequence or for a
protein having both a prosequence and a presequence (leader
sequence).
[0039] The polynucleotides of the present invention may also have
the coding sequence fused in frame to a marker sequence which
allows for purification of the polypeptide of the present
invention. The marker sequence may be a hexa-histidine tag supplied
by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host,
or, for example, the marker sequence may be a hemagglutinin (HA)
tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson, I., et al., Cell, 37:767 (1984)).
[0040] Most highly preferred are nucleic acid molecules encoding
the mature protein having the amino acid sequence shown in SEQ ID
NO:2 as residues 24-331 or 27-331, or the mature NAF-1 amino acid
sequence encoded by the deposited cDNA clone.
[0041] Also highly preferred are nucleic acid molecules encoding
the TSR domain of the protein having the amino acid sequence shown
in SEQ ID NO:9 or the TSR domain of the NAF-1 amino acid sequence
encoded by the deposited cDNA clone.
[0042] Thus, one aspect of the invention provides an isolated
nucleic acid molecule comprising a polynucleotide having a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding a full-length NAF-1 polypeptide having
the complete amino acid sequence in SEQ ID NO:2, or the complete
amino acid sequence encoded by the cDNA clone contained in the ATCC
Deposit No. 97343; (b) a nucleotide sequence encoding a full-length
NAF-1 polypeptide having the complete amino acid sequence in SEQ ID
NO:2 excepting the N-terminal methionine (i.e., positions 2 to 331
of SEQ ID NO:2) or the complete amino acid sequence excepting the
N-terminal methionine encoded by the cDNA clone contained in the
ATCC Deposit No. 97343; (c) a nucleotide sequence encoding a
predicted mature form of the NAF-1 polypeptide having the amino
acid sequence at positions 24-331 or 27-331 in SEQ ID NO:2 or as
encoded by the cDNA clone contained in the ATCC Deposit No. 97343;
(d) a nucleotide sequence encoding a polypeptide comprising the
predicted TSR domain of the NAF-1 polypeptide having the amino acid
sequence at positions 284-330 in SEQ ID NO:2 or as encoded by the
cDNA clone contained in the ATCC Deposit No. 97343; and (e) a
nucleotide sequence complementary to any of the nucleotide
sequences in (a), (b), (c) or (d) above.
[0043] Further embodiments of the invention include isolated
nucleic acid molecules that comprise a polynucleotide having a
nucleotide sequence at least 90% identical, and more preferably at
least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide
sequences in (a), (b), (c), (d) or (e), above, or a polynucleotide
which hybridizes under stringent hybridization conditions to a
polynucleotide in (a), (b), (c), (d) or (e), above. This
polynucleotide which hybridizes does not hybridize under stringent
hybridization conditions to a polynucleotide having a nucleotide
sequence consisting of only A residues or of only T residues. An
additional nucleic acid embodiment of the invention relates to an
isolated nucleic acid molecule comprising a polynucleotide which
encodes the amino acid sequence of an epitope-bearing portion of a
NAF-1 polypeptide having an amino acid sequence in (a), (b), (c),
(d) or (e), above.
[0044] The present invention also relates to recombinant vectors,
which include the isolated nucleic acid molecules of the present
invention, and to host cells containing the recombinant vectors, as
well as to methods of making such vectors and host cells and for
using them for production of NAF-1 polypeptides or peptides by
recombinant techniques.
[0045] By a polynucleotide having a nucleotide sequence at least,
for example, 95% "identical" to a reference nucleotide sequence
encoding a NAF-1 polypeptide is intended that the nucleotide
sequence of the polynucleotide is identical to the reference
sequence except that the polynucleotide sequence may include up to
five point mutations per each 100 nucleotides of the reference
nucleotide sequence encoding the NAF-1 polypeptide. In other words,
to obtain a polynucleotide having a nucleotide sequence at least
95% identical to a reference nucleotide sequence, up to 5% of the
nucleotides in the reference sequence may be deleted or substituted
with another nucleotide, or a number of nucleotides up to 5% of the
total nucleotides in the reference sequence may be inserted into
the reference sequence. These mutations of the reference sequence
may occur at the 5' or 3' terminal positions of the reference
nucleotide sequence or anywhere between those terminal positions,
interspersed either individually among nucleotides in the reference
sequence or in one or more contiguous groups within the reference
sequence.
[0046] As a practical matter, whether any particular nucleic acid
molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to,
for instance, the nucleotide sequence shown in FIG. 1 or to the
nucleotides sequence of the deposited cDNA clone can be determined
conventionally using known computer programs such as the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711). Bestfit uses the local homology
algorithm of Smith and Waterman, Advances in Applied Mathematics
2:482-489 (1981), to find the best segment of homology between two
sequences. When using Bestfit or any other sequence alignment
program to determine whether a particular sequence is, for
instance, 95% identical to a reference sequence according to the
present invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference nucleotide sequence and that gaps in homology of up to 5%
of the total number of nucleotides in the reference sequence are
allowed.
[0047] The present application is directed to nucleic acid
molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1) or to the
nucleic acid sequence of the deposited cDNA, irrespective of
whether they encode a polypeptide having NAF-1 activity. This is
because even where a particular nucleic acid molecule does not
encode a polypeptide having NAF-lactivity, one of skill in the art
would still know how to use the nucleic acid molecule, for
instance, as a hybridization probe or a polymerase chain reaction
(PCR) primer. Uses of the nucleic acid molecules of the present
invention that do not encode a polypeptide having NAF-1 activity
include, inter alia, (1) isolating the NAF-1 gene or allelic
variants thereof in a cDNA library; (2) in situ hybridization
(e.g., "FISH") to metaphase chromosomal spreads to provide precise
chromosomal location of the NAF-1 gene, as described in Verma et
al., Human Chromosomes: A Manual of Basic Techniques, Pergamon
Press, New York (1988); and Northern Blot analysis for detecting
NAF-1 mRNA expression in specific tissues.
[0048] Preferred, however, are nucleic acid molecules having
sequences at least 90%, 95%, 96%, 97%, 98% or 99% identical to the
nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1) or to the
nucleic acid sequence of the deposited cDNA which do, in fact,
encode a polypeptide having NAF-1 protein activity. By "a
polypeptide having NAF-1 activity" is intended polypeptides
exhibiting activity similar, but not necessarily identical, to an
activity of the mature protein of the invention, as measured in a
particular biological assay. For example, the NAF-1 protein of the
present invention causes axonal neurite extension and promotes
neural cell adhesion. Such activity can be assayed as described in
Klar, et al., Cell 69:95-110, incorporated herein by reference.
[0049] NAF-1 protein modulates axonal neurite extension and neural
cell adhesion in a dose-dependent manner in the above-described
assay. Thus, "a polypeptide having NAF-1 protein activity" includes
polypeptides that also exhibit any of the same neurite extension
and neural cell adhesion promoting activities in the
above-described assays in a dose-dependent manner. Although the
degree of dose-dependent activity need not be identical to that of
the NAF-1 protein, preferably, "a polypeptide having NAF-1 protein
activity" will exhibit substantially similar dose-dependence in a
given activity as compared to the NAF-1 protein (i.e., the
candidate polypeptide will exhibit greater activity or not more
than about 25-fold less and, preferably, not more than about
tenfold less activity relative to the reference NAF-1 protein).
[0050] Of course, due to the degeneracy of the genetic code, one of
ordinary skill in the art will immediately recognize that a large
number of the nucleic acid molecules having a sequence at least
90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence of the deposited cDNA or the nucleic acid sequence shown
in FIG. 1 (SEQ ID NO: 1) will encode a polypeptide "having NAF-1
protein activity." In fact, since degenerate variants of these
nucleotide sequences all encode the same polypeptide, this will be
clear to the skilled artisan even without performing the above
described comparison assay. It will be further recognized in the
art that, for such nucleic acid molecules that are not degenerate
variants, a reasonable number will also encode a polypeptide having
NAF-1 protein activity. This is because the skilled artisan is
fully aware of amino acid substitutions that are either less likely
or not likely to significantly effect protein function (e.g.,
replacing one aliphatic amino acid with a second aliphatic amino
acid), as further described below.
[0051] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0052] The present invention is further directed to nucleic acid
molecules encoding portions of the nucleotide sequences described
herein as well as to fragments of the isolated nucleic acid
molecules described herein. In particular, the invention provides a
polynucleotide having a nucleotide sequence representing the
portion of SEQ ID NO:1 which consists of positions 1-1010 of SEQ ID
NO:1.
[0053] In addition, the invention provides nucleic acid molecules
having nucleotide sequences related to extensive portions of SEQ ID
NO:1 which have been determined from the following related cDNA
clones: HLHCE24R (shown as SEQ ID NO: 15); BLHDR83R (shown as SEQ
ID NO:16) and HPTSB36R (shown as SEQ ID NO:17).
[0054] Further, the invention includes a polynucleotide comprising
any portion of at least about 30 nucleotides, preferably at least
about 50 nucleotides, of SEQ ID NO: 1 from residue 1-650.
[0055] More generally, by a fragment of an isolated nucleic acid
molecule having the nucleotide sequence of the deposited cDNA or
the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1) is intended
fragments at least about 15 nt, and more preferably at least about
20 nt, still more preferably at least about 30 nt, and even more
preferably, at least about 40 nt in length which are useful as
diagnostic probes and primers as discussed herein. Of course,
larger fragments 50-300 nt in length are also useful according to
the present invention as are fragments corresponding to most, if
not all, of the nucleotide sequence of the deposited cDNA or as
shown in FIG. 1 (SEQ ID NO:1). By a fragment at least 20 nt in
length, for example, is intended fragments which include 20 or more
contiguous bases from the nucleotide sequence of the deposited cDNA
or the nucleotide sequence as shown in FIG. 1 (SEQ ID NO:1).
Preferred nucleic acid fragments of the present invention include
nucleic acid molecules encoding epitope-bearing portions of the
NAF-1 polypeptide as identified in FIG. 4 and described in more
detail below.
[0056] Fragments of the full length gene of the present invention
may be used as a hybridization probe for a cDNA library to isolate
the full length cDNA and to isolate other cDNAs which have a high
sequence similarity to the gene or similar biological activity.
Probes of this type preferably have at least 30 bases and may
contain, for example, at least 50 bases. The probe may also be used
to identify a cDNA clone corresponding to a full length transcript
and a genomic clone or clones that contain the complete gene
including regulatory and promoter regions, exons, and introns. An
example of a screen comprises isolating the coding region of the
gene by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of the gene of the present invention are used
to screen a library of human cDNA, genomic DNA or mRNA to determine
which members of the library the probe hybridizes to.
[0057] The present invention further relates to polynucleotides
which hybridize to the hereinabove-described sequences if there is
at least 70%, preferably at least 90%, and more preferably at least
95%, 96%, 97%, 98% or 99% identity between the sequences. The
present invention particularly relates to polynucleotides which
hybridize under stringent conditions to the hereinabove-described
polynucleotides. As herein used, the term "stringent hybridization
conditions" is intended overnight incubation at 42.degree. C. in a
solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM
trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA, followed by washing the
filters in 0.1.times.SSC at about 65.degree. C. The polynucleotides
which hybridize to the hereinabove described polynucleotides in a
preferred embodiment encode polypeptides which either retain
substantially the same biological function or activity as the
mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:
1).
[0058] Alternatively, the polynucleotide may have at least 15
bases, preferably at least 30 bases, and more preferably at least
50 bases which hybridize to a polynucleotide of the present
invention and which has an identity thereto, as described, and
which may or may not retain activity. For example, such
polynucleotides may be employed as probes for the polynucleotide of
SEQ ID NO: 1, for example, for recovery of the polynucleotide or as
a diagnostic probe or as a PCR primer.
[0059] Thus, the present invention is directed to polynucleotides
having at least a 70% identity, preferably at least 90% and more
preferably at least a 95%, 96%, 97%, 98%, or 99% identity to a
polynucleotide which encodes the polypeptide of SEQ ID NO:2 and
polynucleotides complementary thereto as well as portions thereof,
which portions have at least 30 consecutive bases and preferably at
least 50 consecutive bases and to polypeptides encoded by such
polynucleotides.
[0060] The present invention further relates to a polypeptide which
has the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2), as
well as fragments, analogs and derivatives of such polypeptide.
[0061] To improve or alter the characteristics of NAF-1
polypeptides, protein engineering may be employed. Recombinant DNA
technology known to those skilled in the art can be used to create
novel mutant proteins or "muteins including single or multiple
amino acid substitutions, deletions, additions or fusion proteins.
Such modified polypeptides can show, e.g., enhanced activity or
increased stability. In addition, they may be purified in higher
yields and show better solubility than the corresponding natural
polypeptide, at least under certain purification and storage
conditions.
[0062] For instance, for many proteins, including the extracellular
domain of a membrane associated protein or the mature form(s) of a
secreted protein, it is known in the art that one or more amino
acids may be deleted from the N-terminus or C-terminus without
substantial loss of biological function. For instance, Ron et al.,
J. Biol. Chem., 268:2984-2988 (1993) reported modified KGF proteins
that had heparin binding activity even if 3, 8, or 27
amino-terminal amino acid residues were missing. In the present
case, since the protein of the invention contains a TSR repeat,
deletions of N-terminal amino acids up to the cysteine at position
284 (C284) of SEQ ID NO:2 may retain some biological activity such
as the ability to promote cell adhesion, however, additional
deletions including C284 would not be expected to retain such
biological activities because it is known that this residue in the
TSR repeat is required for secondary structure necessary to promote
cell adhesion.
[0063] However, even if deletion of one or more amino acids from
the N-terminus of a protein results in modification or loss of one
or more biological functions of the protein, other biological
activities may still be retained. Thus, the ability of the
shortened protein to induce and/or bind to antibodies which
recognize the complete or TSR domain of the protein generally will
be retained when less than the majority of the residues of the
complete or TSR domain are removed from the N-terminus. Whether a
particular polypeptide lacking N-terminal residues of a complete
protein retains such immunologic activities can readily be
determined by routine methods described herein and otherwise known
in the art.
[0064] Accordingly, the present invention further provides
polypeptides having one or more residues deleted from the amino
terminus of the amino acid sequence of the NAF-1 shown in SEQ ID
NO:2, up to the C284 residue, and polynucleotides encoding such
polypeptides. In particular, the present invention provides
polypeptides comprising the amino.
[0065] Similarly, many examples of biologically functional
C-terminal deletion muteins are known. For instance, interferon
gamma shows up to ten times higher activities by deleting 8-10
amino acid residues from the carboxy terminus of the protein
(Dobeli et al., J. Biotechnology 7:199-216 (1988). However, even if
deletion of one or more amino acids from the C-terminus of a
protein results in modification of loss of one or more biological
functions of the protein, other biological activities may still be
retained. Thus, the ability of the shortened protein to induce
and/or bind to antibodies which recognize the complete or TSR
domain of the protein generally will be retained when less than the
majority of the residues of the complete or TSR domain protein are
removed from the C-terminus. Whether a particular polypeptide
lacking C-terminal residues of a complete protein retains such
immunologic activities can readily be determined by routine methods
described herein and otherwise known in the art.
[0066] Accordingly, the present invention further provides
polypeptides having one or more residues from the carboxy terminus
of the amino acid sequence of the NAF-1 shown in SEQ ID NO:2, up to
the C330 residue of SEQ ID NO:2, and polynucleotides encoding such
polypeptides. In particular, the present invention provides
polypeptides having the amino acid sequence of residues 1-m of the
amino acid sequence in SEQ ID NO:2, where m is either 330 or 331,
and C330 is the position of the first residue from the C-terminus
of the complete NAF-1 polypeptide (shown in SEQ ID NO:2) believed
to be required for cell adhesion of the NAF-1 protein.
[0067] The invention also provides polypeptides having one or more
amino acids deleted from both the amino and the carboxyl termini,
which may be described generally as having residues n-m of SEQ ID
NO:2, where n and m are integers as described above.
[0068] Also included are a nucleotide sequence encoding a
polypeptide consisting of a portion of the complete NAF-1 amino
acid sequence encoded by the cDNA clone contained in ATCC Deposit
No. 97343, where this portion excludes from 1 to about 283 amino
acids from the amino terminus of the complete amino acid sequence
encoded by the cDNA clone contained in ATCC Deposit No. 97343, or 1
amino acid from the carboxy terminus, or any combination of the
above amino terminal and carboxy terminal deletions, of the
complete amino acid sequence encoded by the cDNA clone contained in
ATCC Deposit No. 97343. Polynucleotides encoding all of the above
deletion mutant polypeptide forms also are provided.
[0069] In addition to terminal deletion forms of the protein
discussed above, it also will be recognized by one of ordinary
skill in the art that some amino acid sequences of the NAF-1
polypeptide can be varied without significant effect of the
structure or function of the protein. If such differences in
sequence are contemplated, it should be remembered that there will
be critical areas on the protein which determine activity.
[0070] Thus, the invention further includes variations of the NAF-1
polypeptide which show substantial NAF-1 polypeptide activity or
which include regions of NAF-1 protein such as the protein portions
discussed below. Such mutants include deletions, insertions,
inversions, repeats, and type substitutions selected according to
general rules known in the art so as have little effect on
activity. For example, guidance concerning how to make
phenotypically silent amino acid substitutions is provided in
Bowie, J. U. et al., "Deciphering the Message in Protein Sequences:
Tolerance to Amino Acid Substitutions," Science 247:1306-1310
(1990), wherein the authors indicate that there are two main
approaches for studying the tolerance of an amino acid sequence to
change. The first method relies on the process of evolution, in
which mutations are either accepted or rejected by natural
selection. The second approach uses genetic engineering to
introduce amino acid changes at specific positions of a cloned gene
and selections or screens to identify sequences that maintain
functionality.
[0071] As the authors state, these studies have revealed that
proteins are surprisingly tolerant of amino acid substitutions. The
authors further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. For example, most
buried amino acid residues require nonpolar side chains, whereas
few features of surface side chains are generally conserved. Other
such phenotypically silent substitutions are described in Bowie, J.
U. et al., supra, and the references cited therein. Typically seen
as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxyl residues Ser and Thr, exchange of the
acidic residues Asp and Glu, substitution between the amide
residues Asn and Gln, exchange of the basic residues Lys and Arg
and replacements among the aromatic residues Phe, Tyr.
[0072] Thus, the fragment, derivative or analog of the polypeptide
of SEQ ID NO:2, or that encoded by the deposited cDNA, may be (i)
one in which one or more of the amino acid residues are substituted
with a conserved or non-conserved amino acid residue (preferably a
conserved amino acid residue) and such substituted amino acid
residue may or may not be one encoded by the genetic code, or (ii)
one in which one or more of the amino acid residues includes a
substituent group, or (iii) one in which the mature polypeptide is
fused with another compound, such as a compound to increase the
half-life of the polypeptide (for example, polyethylene glycol), or
(iv) one in which the additional amino acids are fused to the above
form of the polypeptide, such as an IgG Fc fusion region peptide or
leader or secretory sequence or a sequence which is employed for
purification of the above form of the polypeptide or a proprotein
sequence. Such fragments, derivatives and analogs are deemed to be
within the scope of those skilled in the art from the teachings
herein
[0073] Thus, the NAF-1 of the present invention may include one or
more amino acid substitutions, deletions or additions, either from
natural mutations or human manipulation. As indicated, changes are
preferably of a minor nature, such as conservative amino acid
substitutions that do not significantly affect the folding or
activity of the protein (see Table 1).
1TABLE 1 Conservative Amino Acid Substitutions. Aromatic
Phenylalanine Tryptophan Tyrosine Hydrophobic Leucine Isoleucine
Valine Polar Glutamine Asparagine Basic Arginine Lysine Histidine
Acidic Aspartic Acid Glutamic Acid Small Alanine Serine Threonine
Methionine Glycine
[0074] Amino acids in the NAF-1 protein of the present invention
that are essential for function can be identified by methods known
in the art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)).
The latter procedure introduces single alanine mutations at every
residue in the molecule. The resulting mutant molecules are then
tested for biological activity such as receptor binding or in vitro
or in vitro proliferative activity.
[0075] Of special interest are substitutions of charged amino acids
with other charged or neutral amino acids which may produce
proteins with highly desirable improved characteristics, such as
less aggregation. Aggregation may not only reduce activity but also
be problematic when preparing pharmaceutical formulations, because
aggregates can be immunogenic (Pinckard et al., Clin. Exp. Immunol.
2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987);
Cleland et al., Crit. Rev. Therapeutic Drug Carrier Systems
10:307.
[0076] FIG. 3 shows the consensus TSR sequence (SEQ ID NO: 14).
Preferred mutants having increased cell adhesion activity are those
with substitutions making the NAF-1 polypeptides more similar to
the consensus sequence.
[0077] The terms "fragment," "derivative" and "analog" when
referring to the polypeptide of FIG. 1 (SEQ ID NO:2), means a
polypeptide which retains essentially the same biological function
or activity as such polypeptide. Thus, an analog includes a
proprotein which can be activated by cleavage of the proprotein
portion to produce an active mature polypeptide.
[0078] The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a synthetic
polypeptide, preferably a recombinant polypeptide.
[0079] The fragment, derivative or analog of the polypeptide of
FIG. 1 (SEQ ID NO:2) may be (i) one in which one or more of the
amino acid residues are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code, or (ii) one in which one or more
of the amino acid residues includes a substituent group, or (iii)
one in which the mature polypeptide is fused with another compound,
such as a compound to increase the half-life of the polypeptide
(for example, polyethylene glycol), or (iv) one in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such fragments, derivatives and analogs are deemed to be within the
scope of those skilled in the art from the teachings herein.
[0080] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0081] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural environment.
[0082] The invention further provides an isolated NAF-1 polypeptide
comprising an amino acid sequence selected from the group
consisting of: (a) the amino acid sequence of the full-length NAF-1
polypeptide having the complete amino acid sequence shown in SEQ ID
NO:2 or the complete amino acid sequence excepting the N-terminal
methionine encoded by the cDNA clone contained in the ATCC Deposit
No. 97343; (b) the amino acid sequence of the full-length NAF-1
polypeptide having the complete amino acid sequence shown in SEQ ID
NO:2 excepting the N-terminal methionine (i.e., positions 1-331 of
SEQ ID NO:2) or the complete amino acid sequence excepting the
N-terminal methionine encoded by the cDNA clone contained in the
ATCC Deposit No. 97343; (c) the amino acid sequence of the mature
NAF-1 polypeptide having the amino acid sequence of residues 24-331
or 27-331 in SEQ ID NO:2, or the mature NAF-1 amino acid sequence
as encoded by the cDNA clone contained in ATCC Deposit No. 97343;
and (d) the amino acid sequence of the TSR domain of NAF-1 having
the amino acid sequence of residues 284 to 330 of SEQ ID NO:2, or
the amino acid sequence of the TSR domain of NAF-1 encoded by the
cDNA clone contained in ATCC Deposit No. 97343.
[0083] Further polypeptides of the present invention include
polypeptides which have at least 90% similarity, more preferably at
least 95% similarity, and still more preferably at least 96%, 97%,
98% or 99% similarity to those described above. The polypeptides of
the invention also comprise those which are at least 80% identical,
more preferably at least 90% or 95% identical, still more
preferably at least 96%, 97%, 98% or 99% identical to the
polypeptide encoded by the deposited cDNA or to the polypeptide of
SEQ ID NO:2, and also include portions of such polypeptides with at
least 30 amino acids and more preferably at least 50 amino
acids.
[0084] By "% similarity" for two polypeptides is intended a
similarity score produced by comparing the amino acid sequences of
the two polypeptides using the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, 575 Science Drive, Madison, Wis. 53711)
and the default settings for determining similarity. Bestfit uses
the local homology algorithm of Smith and Waterman (Advances in
Applied Mathematics 2:482-489, 1981) to find the best segment of
similarity between two sequences.
[0085] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a reference amino acid sequence of a
NAF-1 polypeptide is intended that the amino acid sequence of the
polypeptide is identical to the reference sequence except that the
polypeptide sequence may include up to five amino acid alterations
per each 100 amino acids of the reference amino acid of the NAF-1
polypeptide. In other words, to obtain a polypeptide having an
amino acid sequence at least 95% identical to a reference amino
acid sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0086] As a practical matter, whether any particular polypeptide is
at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance,
the amino acid sequence shown in SEQ ID NO:2 or to the amino acid
sequence encoded by deposited cDNA clone can be determined
conventionally using known computer programs such the Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix,
Genetics Computer Group, University Research Park, 575 Science
Drive, Madison, Wis. 53711). When using Bestfit or any other
sequence alignment program to determine whether a particular
sequence is, for instance, 95% identical to a reference sequence
according to the present invention, the parameters are set, of
course, such that the percentage of identity is calculated over the
full length of the reference amino acid sequence and that gaps in
homology of up to 5% of the total number of amino acid residues in
the reference sequence are allowed.
[0087] In another aspect, the invention provides a peptide or
polypeptide comprising an epitope-bearing portion of a polypeptide
of the invention. The epitope of this polypeptide portion is an
immunogenic or antigenic epitope of a polypeptide of the invention.
An "immunogenic epitope" is defined as a part of a protein that
elicits an antibody response when the whole protein is the
immunogen. On the other hand, a region of a protein molecule to
which an antibody can bind is defined as an "antigenic epitope."
The number of immunogenic epitopes of a protein generally is less
than the number of antigenic epitopes. See, for instance, Geysen et
al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).
[0088] As to the selection of peptides or polypeptides bearing an
antigenic epitope (i.e., that contain a region of a protein
molecule to which an antibody can bind), it is well known in that
art that relatively short synthetic peptides that mimic part of a
protein sequence are routinely capable of eliciting an antiserum
that reacts with the partially mimicked protein. See, for instance,
Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A.
(1983) "Antibodies that react with predetermined sites on
proteins," Science, 219:660-666. Peptides capable of eliciting
protein-reactive sera are frequently represented in the primary
sequence of a protein, can be characterized by a set of simple
chemical rules, and are confined neither to immunodominant regions
of intact proteins (i.e., immunogenic epitopes) nor to the amino or
carboxyl terminals. Antigenic epitope-bearing peptides and
polypeptides of the invention are therefore useful to raise
antibodies, including monoclonal antibodies, that bind specifically
to a polypeptide of the invention. See, for instance, Wilson et
al., Cell 37:767-778 (1984) at 777.
[0089] Antigenic epitope-bearing peptides and polypeptides of the
invention preferably contain a sequence of at least seven, more
preferably at least nine and most preferably between about 15 to
about 30 amino acids contained within the amino acid sequence of a
polypeptide of the invention. Non-limiting examples of antigenic
polypeptides or peptides that can be used to generate
NAF-1-specific antibodies include: a polypeptide comprising amino
acid residues from about Ala-75 to about Arg-100; a polypeptide
comprising amino acid residues from about Leu-168 to about Ala-180;
a polypeptide comprising amino acid residues from about Thr-204 to
about Tyr-226; a polypeptide comprising amino acid residues from
about-Leu-258 to about Ser-281; and a polypeptide comprising amino
acid residues from about Gly-291 to about Pro-327. These
polypeptide fragments have been determined to bear antigenic
epitopes of the NAF-1 protein by the analysis of the Jameson-Wolf
antigenic index, as shown in FIG. 4, above.
[0090] The epitope-bearing peptides and polypeptides of the
invention may be produced by any conventional means See, e.g.,
Houghten, R. A. (1985) "General method for the rapid solid-phase
synthesis of large numbers of peptides: specificity of
antigen-antibody interaction at the level of individual amino
acids." Proc. Natl. Acad. Sci. USA 82:5131-5135; this "Simultaneous
Multiple Peptide Synthesis (SMPS)" process is further described in
U.S. Pat. No. 4,631,211 to Houghten et al. (1986).
[0091] Epitope-bearing peptides and polypeptides of the invention
are used to induce antibodies according to methods well known in
the art. See, for instance, Sutcliffe et al., supra; Wilson et al.,
supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA 82:910-914; and
Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985).
Immunogenic epitope-bearing peptides of the invention, i.e., those
parts of a protein that elicit an antibody response when the whole
protein is the immunogen, are identified according to methods known
in the art. See, for instance, Geysen et al., supra. Further still,
U.S. Pat. No. 5,194,392 to Geysen (1990) describes a general method
of detecting or determining the sequence of monomers (amino acids
or other compounds) which is a topological equivalent of the
epitope (i.e., a "mimotope") which is complementary to a particular
paratope (antigen binding site) of an antibody of interest. More
generally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a
method of detecting or determining a sequence of monomers which is
a topographical equivalent of a ligand which is complementary to
the ligand binding site of a particular receptor of interest.
Similarly, U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996)
on Peralkylated Oligopeptide Mixtures discloses linear
C.sub.1-C.sub.7-alkyl peralkylated oligopeptides and sets and
libraries of such peptides, as well as methods for using such
oligopeptide sets and libraries for determining the sequence of a
peralkylated oligopeptide that preferentially binds to an acceptor
molecule of interest. Thus, non-peptide analogs of the
epitope-bearing peptides of the invention also can be made
routinely by these methods.
[0092] Fragments or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to synthesize
full-length polynucleotides of the present invention.
[0093] The present invention also relates to vectors which include
polynucleotides of the present invention, host cells which are
genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques.
[0094] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the present invention. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0095] The polynucleotides of the present invention may be employed
for producing polypeptides by recombinant techniques. Thus, for
example, the polynucleotide may be included in any one of a variety
of expression vectors for expressing a polypeptide. Such vectors
include chromosomal, nonchromosomal and synthetic DNA sequences,
e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of
plasmids and phage DNA, viral DNA such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies. However, any other vector may be
used as long as it is replicable and viable in the host.
[0096] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0097] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage lambda P.sub.L promoter and other
promoters known to control expression of genes in prokaryotic or
eukaryotic cells or their viruses. The expression vector also
contains a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0098] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0099] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the protein.
[0100] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; plant cells, etc. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0101] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,
phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a, pNH18A,
pNH46A (Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0102] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda P.sub.R, P.sub.L, and trp. Eukaryotic promoters include
CMV immediate early, HSV thymidine kinase, early and late SV40,
LTRs from retrovirus, and mouse metallothionein-I. Selection of the
appropriate vector and promoter is well within the level of
ordinary skill in the art.
[0103] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0104] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0105] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
[0106] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0107] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), a-factor, acid phosphatase, or heat shock proteins,
among others. The heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can
encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product.
[0108] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0109] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0110] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0111] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0112] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well known to those skilled in the art.
[0113] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0114] The polypeptide can be recovered and purified from
recombinant cell cultures by methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used, as necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0115] The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
[0116] NAF-1 may be employed to treat spinal cord injuries or
damage to peripheral nerves by increasing spinal cord and sensory
neuron attachment and neurite outgrowth.
[0117] NAF-1 may also be employed to inhibit tumor cell metastases
induced by small cell carcinoma. The NAF-1 gene and gene product of
the present invention may also be employed to reduce primary tumor
growth, metastatic potential and angiogenesis in human breast
carcinoma cells.
[0118] The NAF-1 gene and gene product of the present invention may
also be employed to promote wound healing due to its ability to
promote cell-cell interaction and cell adhesion.
[0119] NAF-1 may also be employed to modulate hemostasis.
[0120] The polynucleotides and polypeptides of the present
invention may be employed as research reagents and materials for
discovery of treatments and diagnostics to human disease.
[0121] This invention provides a method for identification of the
receptor for NAF-1. The gene encoding methods known to those of
skill in the art, for example, ligand panning and FACS sorting
(Coligan, et al., Current Protocols in Immun., 1(2), Chapter 5,
(1991)). Preferably, expression cloning is employed wherein
polyadenylated RNA is prepared from a cell responsive to NAF-1, and
a cDNA library created from this RNA is divided into pools and used
to transfect COS cells or other cells that are not responsive to
NAF-1. Transfected cells which are grown on glass slides are
exposed to labeled NAF-1 ligand. NAF-1 can be labeled by a variety
of means including iodination or inclusion of a recognition site
for a site-specific protein kinase. Following fixation and
incubation, the slides are subjected to auto-radiographic analysis.
Positive pools are identified and sub-pools are prepared and
re-transfected using an iterative sub-pooling and re-screening
process, eventually yielding a single clone that encodes the
putative receptor. As an alternative approach for receptor
identification, labeled ligand can be photoaffinity linked with
cell membrane or extract preparations that express the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to
X-ray film. The labeled complex containing the ligand-receptor can
be excised, resolved into peptide fragments, and subjected to
protein microsequencing. The amino acid sequence obtained from
microsequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
[0122] This invention provides a method of screening compounds to
identify those which are agonists to or antagonists to NAF-1. The
identification of both type compounds would involve a neurite
outgrowth assay. COS cells (5.times.10.sup.8) are transfected with
NAF-1/pcDNA-1 (Invitrogen, Inc.) and conditioned medium is
collected. NAF.sup.myc is affinity purified on a monoclonal
anti-myc (9E10) affinity-purified F-spondin.sup.myc (20 mg/ml) is
absorbed onto nitrocellulose (Lemmon et al., 1989). For controls,
parental COS cell-conditioned medium is purified on the same column
and used as a substrate on nitrocellulose. The nitrocellulose is
then blocked with BSA (10 mg/ml), which provided a further control
for background neurite outgrowth. Rat E14 DRG neurons are plated on
immobilized protein substrates at a density of 2-10.times.10.sup.4
cells per 35 mm tissue culture dish (Nunc) and grown for 14 hr.
Cultures are then fixed in 4% paraformaldehyde, permeabilized with
0.1% Triton X-100, and stained using MAb 3A10 (Furley et al., 1990;
available from Developmental Studies Hybridoma Bank), which
recognizes a neuronal filament-associated protein and serves as a
marker for fine neurites. Neuronal cell bodies and neurites are
visualized by indirect immunofluorescence on a Zeiss Axioplan
microscope. Neurite lengths are measured as the distance from the
edge of the soma (sharply defined by 3A10 fluorescence) to the tip
of its longest neurite. Neurite lengths are measured if the entire
length of the neurite could be unambiguously identified. About 25
neurites are measurable within each protein-coated area (3-4
mm.sup.2).
[0123] Rat el3 dorsal spinal cord neurons can also be assayed by
plating the dissociated cells on immobilized protein substrate at a
density of 10.sup.6 cells per 35 mm tissue culture dish (Nunc).
After 1 hr. the cultures are washed twice with PBS and fixed in 4%
paraformaldehyde. Cells are counted on a Zeiss Axioplan microscope
at 400.times. magnification. Ten independent counts are taken from
each experiment.
[0124] An alternative example of identifying agonists and
antagonists to the polypeptide of the present invention includes
expressing the NAF-1 receptor from a mammalian cell or membrane
preparation and incubating that receptor with labeled NAF-1 in the
presence of a compound. The ability of a compound to enhance or
block the interaction is then quantified. Alternatively, the
response of a known second messenger system following interaction
of NAF-1 and its receptor would be measured and compared in the
presence or absence of the compound. Such second messenger systems
include, but are not limited, cAMP guanylate cyclase, ion channels
or phosphoinositide hydrolysis.
[0125] Potential antagonists include an antibody, or in some cases,
an oligopeptide, which binds to the polypeptide. Alternatively, a
potential antagonist may be a closely related protein which binds
to the receptor sites, however, they are inactive forms of the
polypeptide and thereby prevent the action of NAF-1 since receptor
sites are occupied.
[0126] Another potential antagonist is an antisense construct
prepared using antisense technology. Antisense technology can be
used to control gene expression through triple-helix formation or
antisense DNA or RNA, both of which methods are based on binding of
a polynucleotide to DNA or RNA. For example, the 5' coding portion
of the polynucleotide sequence, which encodes for the mature
polypeptides of the present invention, is used to design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in
length. A DNA oligonucleotide is designed to be complementary to a
region of the gene involved in transcription (triple helix --see
Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science,
241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),
thereby preventing transcription and the production of NAF-1. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into NAF-1 polypeptide
(Antisense--Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of NAF-1.
[0127] Potential antagonists include a small molecule which binds
to and occupies the catalytic site of the polypeptide thereby
making the catalytic site inaccessible to substrate such that
normal biological activity is prevented. Examples of small
molecules include but are not limited to small peptides or
peptide-like molecules.
[0128] The antagonists may be employed to treat malarial infection
induced by Plasmodium falciparum. During malarial infection, the
polypeptide of the present invention may promote adhesion of
parasitized red cells to endothelial cells and, therefore,
antagonists would inhibit this action and prevent malaria. The
antagonists may also be employed to treat cancer, for example, in
blocking metastasis by inhibiting cell adhesion.
[0129] The polypeptides of the present invention or antagonists and
agonists may be employed in combination with a suitable
pharmaceutical carrier. Such compositions comprise a
therapeutically effective amount of the polypeptide or antagonists
or agonist, and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The formulation should suit the mode of
administration.
[0130] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides of the present
invention or agonists or antagonists may be employed in conjunction
with other therapeutic compounds.
[0131] The pharmaceutical compositions may be administered in a
convenient manner such as by the oral, topical, parenterally,
intravenous, intraperitoneal, intramuscular, subcutaneous,
intranasal or intradermal routes. The pharmaceutical compositions
are administered in an amount which is effective for treating
and/or prophylaxis of the specific indication. In general, they are
administered in an amount of at least about 10 .mu.g/kg body weight
and in most cases they will be administered in an amount not in
excess of about 8 mg/Kg body weight per day. In most cases, the
dosage is from about 10 .mu.g/kg to about 1 mg/kg body weight
daily, taking into account the routes of administration, symptoms,
etc.
[0132] The NAF-1 polypeptides and agonists and antagonists which
are polypeptides may also be employed in accordance with the
present invention by expression of such polypeptides in vivo, which
is often referred to as "gene therapy."
[0133] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo,
with the engineered cells then being provided to a patient to be
treated with the polypeptide. Such methods are well-known in the
art and are apparent from the teachings herein. For example, cells
may be engineered by the use of a retroviral plasmid vector
containing RNA encoding a polypeptide of the present invention.
[0134] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by, for example, procedures known in the art.
For example, a packaging cell is transduced with a retroviral
plasmid vector containing RNA encoding a polypeptide of the present
invention such that the packaging cell now produces infectious
viral particles containing the gene of interest. These producer
cells may be administered to a patient for engineering cells in
vivo and expression of the polypeptide in vivo. These and other
methods for administering a polypeptide of the present invention by
such method should be apparent to those skilled in the art from the
teachings of the present invention.
[0135] Retroviruses from which the retroviral plasmid vectors
hereinabove mentioned may be derived include, but are not limited
to, Moloney Murine Leukemia Virus, spleen necrosis virus,
retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,
avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus. In one embodiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
[0136] The vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited to,
the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus (CMV) promoter described in Miller, et al.,
Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter
(e.g., cellular promoters such as eukaryotic cellular promoters
including, but not limited to, the histone, pol III, and b-actin
promoters). Other viral promoters which may be employed include,
but are not limited to, adenovirus promoters, thymidine kinase (TK)
promoters, and B19 parvovirus promoters. The selection of a
suitable promoter will be apparent to those skilled in the art from
the teachings contained herein.
[0137] The nucleic acid sequence encoding the polypeptide of the
present invention is under the control of a suitable promoter.
Suitable promoters which may be employed include, but are not
limited to, adenoviral promoters, such as the adenoviral major late
promoter; or heterologous promoters, such as the cytomegalovirus
(CMV) promoter; the respiratory syncytial virus (RSV) promoter;
inducible promoters, such as the MMT promoter, the metallothionein
promoter; heat shock promoters; the albumin promoter; the ApoAI
promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral
LTRs (including the modified retroviral LTRs hereinabove
described); the b-actin promoter; and human growth hormone
promoters. The promoter also may be the native promoter which
controls the gene encoding the polypeptide.
[0138] The retroviral plasmid vector is employed to transduce
packaging cell lines to form producer cell lines. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, y-2, y-AM, PA12, T19-14.times.,
VT-19-17-H2, yCRE, yCRIP, GP+E-86, GP+envAm12, and DAN cell lines
as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14
(1990), which is incorporated herein by reference in its entirety.
The vector may transduce the packaging cells through any means
known in the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and CaPO.sub.4
precipitation. In one alternative, the retroviral plasmid vector
may be encapsulated into a liposome, or coupled to a lipid, and
then administered to a host.
[0139] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed, to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) encoding the polypeptide. Eukaryotic cells which
may be transduced include, but are not limited to, embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
[0140] This invention is also related to the use of the gene of the
present invention as a diagnostic. Detection of a mutated form of
the gene will allow a diagnosis of a disease or a susceptibility to
a disease which results from underexpression of NAF-1, for example,
tumor metastases and tumor angiogenesis.
[0141] Individuals carrying mutations in the gene of the present
invention may be detected at the DNA level by a variety of
techniques. Nucleic acids for diagnosis may be obtained from a
patient's cells, including but not limited to blood, urine, saliva,
tissue biopsy and autopsy material. The genomic DNA may be used
directly for detection or may be amplified enzymatically by using
PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.
RNA or cDNA may also be used for the same purpose. As an example,
PCR primers complementary to the nucleic acid encoding NAF-1 can be
used to identify and analyze mutations. For example, deletions and
insertions can be detected by a change in size of the amplified
product in comparison to the normal genotype. Point mutations can
be identified by hybridizing amplified DNA to radiolabeled RNA or
alternatively, radiolabeled antisense DNA sequences. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase A digestion or by differences in melting temperatures.
[0142] Sequence differences between the reference gene and genes
having mutations may be revealed by the direct DNA sequencing
method. In addition, cloned DNA segments may be employed as probes
to detect specific DNA segments. The sensitivity of this method is
greatly enhanced when combined with PCR. For example, a sequencing
primer is used with double-stranded PCR product or a
single-stranded template molecule generated by a modified PCR. The
sequence determination is performed by conventional procedures with
radiolabeled nucleotide or by automatic sequencing procedures with
fluorescent-tags.
[0143] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments in gels with or without denaturing agents. Small
sequence deletions and insertions can be visualized by high
resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science, 230:1242 (1985)).
[0144] Sequence changes at specific locations may also be revealed
by nuclease protection assays, such as RNase and S1 protection or
the chemical cleavage method (e.g., Cotton et al., PNAS, USA,
85:4397-4401 (1985)).
[0145] Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing or the use of restriction
enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP))
and Southern blotting of genomic DNA.
[0146] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations can also be detected by in situ analysis.
[0147] The present invention also relates to a diagnostic assay for
detecting altered levels of the polypeptide of the present
invention in various tissues since an over-expression of the
proteins compared to normal control tissue samples can detect the
presence of NAF-1 and conditions related to an overexpression of
NAF-1, for example, tumor metastases and angiogenesis. Assays used
to detect levels of the polypeptide of the present invention in a
sample derived from a host are well-known to those of skill in the
art and include radioimmunoassays, competitive-binding assays,
Western Blot analysis and preferably an ELISA assay. An ELISA assay
initially comprises preparing an antibody specific to the NAF-1
antigen, preferably a monoclonal antibody. In addition a reporter
antibody is prepared against the monoclonal antibody. To the
reporter antibody is attached a detectable reagent such as
radioactivity, fluorescence or in this example a horseradish
peroxidase enzyme. A sample is now removed from a host and
incubated on a solid support, e.g. a polystyrene dish, that binds
the proteins in the sample. Any free protein binding sites on the
dish are then covered by incubating with a non-specific protein
such as bovine serum albumin. Next, the monoclonal antibody is
incubated in the dish during which time the monoclonal antibodies
attached to any of the polypeptide of the present invention
attached to the polystyrene dish. All unbound monoclonal antibody
is washed out with buffer. The reporter antibody linked to
horseradish peroxidase is now placed in the dish resulting in
binding of the reporter antibody to any monoclonal antibody bound
to the polypeptide of the present invention. Unattached reporter
antibody is then washed out. Peroxidase substrates are then added
to the dish and the amount of color developed in a given time
period is a measurement of the amount of the polypeptide of the
present invention present in a given volume of patient sample when
compared against a standard curve.
[0148] A competition assay may be employed wherein antibodies
specific to the polypeptide of the present invention are attached
to a solid support and labeled NAF-1 and a sample derived from the
host are passed over the solid support and the amount of label
detected attached to the solid support can be correlated to a
quantity of the polypeptide of the present invention in the
sample.
[0149] The sequences of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to
and can hybridize with a particular location on an individual human
chromosome. Moreover, there is a current need for identifying
particular sites on the chromosome. Few chromosome marking reagents
based on actual sequence data (repeat polymorphisms) are presently
available for marking chromosomal location. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0150] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the 3' untranslated region of the gene is used to rapidly select
primers that do not span more than one exon in the genomic DNA,
thus complicating the amplification process. These primers are then
used for PCR screening of somatic cell hybrids containing
individual human chromosomes. Only those hybrids containing the
human gene corresponding to the primer will yield an amplified
fragment.
[0151] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0152] Fluorescence in situ hybridization (FISH) of a cDNA clone to
a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA having at least 50 or 60 bases. For a review of this
technique, see Verma et al., Human Chromosomes: a Manual of Basic
Techniques, Pergamon Press, New York (1988).
[0153] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man (available on
line through Johns Hopkins University Welch Medical Library). The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0154] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0155] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0156] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0157] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptides itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native polypeptides.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
[0158] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, 1975, Nature, 256:495-497), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983, Immunology
Today 4:72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0159] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice may be used to express humanized
antibodies to immunogenic polypeptide products of this
invention.
[0160] The above-described antibodies may be employed to isolate or
to identify clones expressing the polypeptide or to purify the
polypeptide of the present invention by attachment of the antibody
to a solid support for isolation and/or purification by affinity
chromatography.
[0161] The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified, are by
weight.
[0162] In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms will be
described.
[0163] "Plasmids" are designated by a lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
[0164] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 mg of plasmid
or DNA fragment is used with about 2 units of enzyme in about 20
mil of buffer solution. For the purpose of isolating DNA fragments
for plasmid construction, typically 5 to 50 mg of DNA are digested
with 20 to 250 units of enzyme in a larger volume. Appropriate
buffers and substrate amounts for particular restriction enzymes
are specified by the manufacturer. Incubation times of about 1 hour
at 37.degree. C. are ordinarily used, but may vary in accordance
with the supplier's instructions. After digestion the reaction is
electrophoresed directly on a polyacrylamide gel to isolate the
desired fragment.
[0165] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel, D. et al.,
Nucleic Acids Res., 8:4057 (1980).
[0166] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0167] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T., et al., Id., p. 146). Unless otherwise provided, ligation may
be accomplished using known buffers and conditions with 10 units of
T4 DNA ligase ("ligase") per 0.5 mg of approximately equimolar
amounts of the DNA fragments to be ligated. Unless otherwise
stated, transformation was performed as described in the method of
Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).
EXAMPLES
Example 1
[0168] Bacterial Expression and Purification of NAF-1
[0169] The DNA sequence encoding NAF-1, ATCC #97343, is initially
amplified using PCR oligonucleotide primers corresponding to the 5'
sequences of the processed NAF-1 protein (minus the signal peptide
sequence) and the vector sequences 3' to the NAF-1 gene. Additional
nucleotides corresponding to NAF-1 are added to the 5' and 3'
sequences respectively. The 5' oligonucleotide primer has the
sequence 5' GCCATACGGGATCCCAGCCTCTTGGGGGAGAGTCC 3' (SEQ ID NO:3)
contains a BamHI restriction enzyme site followed by 21 nucleotides
of NAF-1 coding sequence starting from the presumed terminal amino
acid of the processed protein codon. The 3' sequence 5'
GGCATACGTCTAGATTAGACGCAGTTATCAGGGAC 3' (SEQ ID NO:4) contains
complementary sequences to an XbaI site and is followed by 21
nucleotides of NAF-1. The restriction enzyme sites correspond to
the restriction enzyme sites on the bacterial expression vector
pQE-9 (Qiagen, Inc. Chatsworth, Calif.). pQE-9 encodes antibiotic
resistance (Amp.sup.r), a bacterial origin of replication (ori), an
IPTG-regulatable promoter operator (P/O), a ribosome binding site
(RBS), a 6-His tag and restriction enzyme sites. pQE-9 is then
digested with BamHI and XbaI. The amplified sequences are ligated
into pQE-9 and are inserted in frame with the sequence encoding for
the histidine tag and the RBS. The ligation mixture is then used to
transform E. coli strain M15/rep 4 (Qiagen, Inc.) by the procedure
described in Sambrook, J. et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains
multiple copies of the plasmid pREP4, which expresses the lacI
repressor and also confers kanamycin resistance (Kan.sup.r).
Transformants are identified by their ability to grow on LB plates
and ampicillin/kanamycin resistant colonies are selected. Plasmid
DNA is isolated and confirmed by restriction analysis. Clones
containing the desired constructs are grown overnight (O/N) in
liquid culture in LB media supplemented with both Amp (100 ug/ml)
and Kan (25 ug/ml). The O/N culture is used to inoculate a large
culture at a ratio of 1:100 to 1:250. The cells are grown to an
optical density 600 (O.D..sup.600) of between 0.4 and 0.6. IPTG
("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final
concentration of 1 mM. IPTG induces by inactivating the lacI
repressor, clearing the P/O leading to increased gene expression.
Cells are grown an extra 3 to 4 hours. Cells are then harvested by
centrifugation. The cell pellet is solubilized in the chaotropic
agent 6 Molar Guanidine HCl. After clarification, solubilized NAF-1
is purified from this solution by chromatography on a
Nickel-Chelate column under conditions that allow for tight binding
by proteins containing the 6-His tag. NAF-1 is eluted from the
column in 6 molar guanidine HCl pH 5.0 and for the purpose of
renaturation adjusted to 3 molar guanidine HCl, 100 mM sodium
phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione
(oxidized). After incubation in this solution for 12 hours the
protein is dialyzed to 10 mmolar sodium phosphate.
Example 2
[0170] Cloning and Expression of NAF-1 Using the Baculovirus
Expression System
[0171] The DNA sequence encoding the full length NAF-1 protein,
ATCC #97343, was amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene:
[0172] The 5' primer has the sequence 5' GCCATACGGGATCCGCCATCATGG
AAAACCCCAGCCCGGCC 3' (SEQ ID NO:5) and contains a BamHI restriction
enzyme site (in bold) followed by 8 nucleotides resembling an
efficient signal for the initiation of translation in eukaryotic
cells (Kozak, M., J. Mol. Biol., 196:947-950 (1987) which is just
behind the first 21 nucleotides of the NAF-1 gene (the initiation
codon for translation "ATG" is underlined).
[0173] The 3' primer has the sequence 5' GGCATACGTCTAGATTAGACGCAGT
TATCAGGGAC 3' (SEQ ID NO:6) and contains the cleavage site for the
restriction endonuclease XbaI and 21 nucleotides complementary to
the 3' end of the translated sequence of the NAF-1 gene. The
amplified sequences were isolated from a 1% agarose gel using a
commercially available kit ("Geneclean," BIO 101 Inc., La Jolla,
Calif.). The fragment was then digested with the endonucleases
BamHI and XbaI and then purified again on a 1% agarose gel. This
fragment is designated F2.
[0174] The vector pRG1 (modification of pVL941 vector, discussed
below) is used for the expression of the NAF-1 protein using the
baculovirus expression system (for review see: Summers, M. D. and
Smith, G. E. 1987, A manual of methods for baculovirus vectors and
insect cell culture procedures, Texas Agricultural Experimental
Station Bulletin No. 1555). This expression vector contains the
strong polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by the recognition sites for
the restriction endonucleases BamHI and XbaI. The polyadenylation
site of the simian virus (SV)40 is used for efficient
polyadenylation. For an easy selection of recombinant virus the
beta-galactosidase gene from E. coli is inserted in the same
orientation as the polyhedrin promoter followed by the
polyadenylation signal of the polyhedrin gene. The polyhedrin
sequences are flanked at both sides by viral sequences for the
cell-mediated homologous recombination of co-transfected wild-type
viral DNA. Many other baculovirus vectors could be used in place of
pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers,
M. D., Virology, 170:31-39).
[0175] The plasmid was digested with the restriction enzymes BamHI
and XbaI and then dephosphorylated using calf intestinal
phosphatase by procedures known in the art. The DNA was then
isolated from a 1% agarose gel using the commercially available kit
("Geneclean" BIO 101 Inc., La Jolla, Calif.). This vector DNA is
designated V2.
[0176] Fragment F2 and the dephosphorylated plasmid V2 were ligated
with T4 DNA ligase. E. coli XL1 blue cells were then transformed
and bacteria identified that contained the plasmid (pBacNAF-1) with
the NAF-1 gene using the enzymes BamHI and XbaI. The sequence of
the cloned fragment was confirmed by DNA sequencing.
[0177] 5 mg of the plasmid pBacNAF-1 was co-transfected with 1.0 mg
of a commercially available linearized baculovirus (BaculoGold.TM.
baculovirus DNA, Pharminogen, San Diego, Calif.) using the
lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA,
84:7413-7417 (1987)).
[0178] 1 mg of BaculoGold.TM. virus DNA and 5 mg of the plasmid
pBacNAF-1 were mixed in a sterile well of a microtiter plate
containing 50 ml of serum free Grace's medium (Life Technologies
Inc., Gaithersburg, Md.). Afterwards 10 ml Lipofectin plus 90 ml
Grace's medium were added, mixed and incubated for 15 minutes at
room temperature. Then the transfection mixture was added drop-wise
to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue
culture plate with 1 ml Grace's medium without serum. The plate was
rocked back and forth to mix the newly added solution. The plate
was then incubated for 5 hours at 27.degree. C. After 5 hours the
transfection solution was removed from the plate and 1 ml of
Grace's insect medium supplemented with 10% fetal calf serum was
added. The plate was put back into an incubator and cultivation
continued at 27.degree. C. for four days.
[0179] After four days the supernatant was collected and a plaque
assay performed similar as described by Summers and Smith (supra).
As a modification an agarose gel with "Blue Gal" (Life Technologies
Inc., Gaithersburg) was used which allows an easy isolation of blue
stained plaques. (A detailed description of a "plaque assay" can
also be found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0180] Four days after the serial dilution the virus was added to
the cells and blue stained plaques were picked with the tip of an
Eppendorf pipette. The agar containing the recombinant viruses was
then resuspended in an Eppendorf tube containing 200 ml of Grace's
medium. The agar was removed by a brief centrifugation and the
supernatant containing the recombinant baculovirus was used to
infect Sf9 cells seeded in 35 mm dishes. Four days later the
supernatants of these culture dishes were harvested and then stored
at 4.degree. C.
[0181] Sf9 cells were grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells were infected with the recombinant
baculovirus V-NAF-1 at a multiplicity of infection (MOI) of 2. Six
hours later the medium was removed and replaced with SF900 II
medium minus methionine and cysteine (Life Technologies Inc.,
Gaithersburg). 42 hours later 5 mCi of .sup.35S-methionine and 5
mCi .sup.35S cysteine (Amersham) were added. The cells were further
incubated for 16 hours before they were harvested by centrifugation
and the labeled proteins visualized by SDS-PAGE and
autoradiography.
Example 3
[0182] Expression of Recombinant NAF-1 in COS Cells
[0183] Expression of plasmid, NAF-1 HA is derived from a vector
pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication,
2) ampicillin resistance gene, 3) E. coli replication origin, 4)
CMV promoter followed by a polylinker region, an SV40 intron and
polyadenylation site. A DNA fragment encoding the entire NAF-1
precursor and a HA tag fused in frame to its 3' end is cloned into
the polylinker region of the vector, placing the recombinant
protein expression under control of the CMV promoter. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein as previously described (I. Wilson, H. Niman, R. Heighten,
A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767,
(1984)). The fusion of HA tag to the NAF-1 protein allows easy
detection of the recombinant protein with an antibody that
recognizes the HA epitope.
[0184] The plasmid construction strategy is described as
follows:
[0185] The DNA sequence encoding NAF-1, ATCC #97343, is constructed
by PCR using two primers as described in the above examples. The 5'
primer contains a convenient restriction site followed by a portion
of NAF-1 coding sequence starting from the initiation codon; the 3'
sequence contains complementary sequences to a convenient
restriction site, translation stop codon, HA tag and the last
several nucleotides of the NAF-1 coding sequence (not including the
stop codon). Therefore, the PCR product contains a convenient 5'
and 3' restriction sites, NAF-1 coding sequence followed by HA tag
fused in frame, and a translation termination stop codon next to
the HA tag. The PCR amplified DNA fragment and the vector,
pcDNAI/Amp, are digested and ligated. The ligation mixture is
transformed into E. coli strain SURE (available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif.
92037) the transformed culture is plated on ampicillin media plates
and resistant colonies are selected. Plasmid DNA is isolated from
transformants and examined by restriction analysis for the presence
of the correct fragment. For expression of the recombinant NAF-1,
COS cells are transfected with the expression vector by
DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis,
Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory
Press, (1989)). The expression of the NAF-1 HA protein is detected
by radiolabeling and immunoprecipitation method (E. Harlow, D.
Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, (1988)). Cells are labeled for 8 hours with
.sup.35S-cysteine two days post transfection. Culture media is then
collected and cells were lysed with detergent (RIPA buffer (150 mM
NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)
(Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and
culture media are precipitated with an HA specific monoclonal
antibody. Proteins precipitated are analyzed on 15% SDS-PAGE
gels.
Example 4
[0186] Expression via Gene Therapy
[0187] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in tissue-culture medium and separated
into small pieces. Small chunks of the tissue are placed on a wet
surface of a tissue culture flask, approximately ten pieces are
placed in each flask. The flask is turned upside down, closed tight
and left at room temperature over night. After 24 hours at room
temperature, the flask is inverted and the chunks of tissue remain
fixed to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10% FBS, penicillin and streptomycin, is added. This is
then incubated at 37.degree. C. for approximately one week. At this
time, fresh media is added and subsequently changed every several
days. After an additional two weeks in culture, a monolayer of
fibroblasts emerge. The monolayer is trypsinized and scaled into
larger flasks.
[0188] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988)
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[0189] The cDNA encoding a polypeptide of the present invention is
amplified using PCR primers which correspond to the 5' and 3' end
sequences respectively. The 5' primer containing an EcoRI site and
the 3' primer further includes a HindIII site. Equal quantities of
the Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is used to transform bacteria HB101, which are then plated onto
agar-containing kanamycin for the purpose of confirming that the
vector had the gene of interest properly inserted.
[0190] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the gene is then added to
the media and the packaging cells are transduced with the vector.
The packaging cells now produce infectious viral particles
containing the gene (the packaging cells are now referred to as
producer cells).
[0191] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his.
[0192] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product.
[0193] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, within the scope of the appended claims, the invention
may be practiced otherwise than as particularly described.
Sequence CWU 1
1
18 1 1105 DNA Homo sapiens CDS (19)..(1011) 1 cgctgctcct gccgggtg
atg gaa aac ccc agc ccg gcc gcc gcc ctg ggc 51 Met Glu Asn Pro Ser
Pro Ala Ala Ala Leu Gly 1 5 10 aag gcc ctc tgc gct ctc ctc ctg gcc
act ctc ggc gcc gcc ggc cag 99 Lys Ala Leu Cys Ala Leu Leu Leu Ala
Thr Leu Gly Ala Ala Gly Gln 15 20 25 cct ctt ggg gga gag tcc atc
tgt tcc gcc aga gcc ctg gcc aaa tac 147 Pro Leu Gly Gly Glu Ser Ile
Cys Ser Ala Arg Ala Leu Ala Lys Tyr 30 35 40 agc atc acc ttc acg
ggc aag tgg agc cag acg gcc ttc ccc aag cag 195 Ser Ile Thr Phe Thr
Gly Lys Trp Ser Gln Thr Ala Phe Pro Lys Gln 45 50 55 tac ccc ctg
ttc cgc ccc cct gcc cag tgg tct tcg ctg ctg ggg gcc 243 Tyr Pro Leu
Phe Arg Pro Pro Ala Gln Trp Ser Ser Leu Leu Gly Ala 60 65 70 75 gcg
cat agc tcc gac tac agc atg tgg agg aag aac cag tac gtc agt 291 Ala
His Ser Ser Asp Tyr Ser Met Trp Arg Lys Asn Gln Tyr Val Ser 80 85
90 aac ggg ctg cgc gac ttt gcg gag cgc ggc gag gcc tgg gcg ctg atg
339 Asn Gly Leu Arg Asp Phe Ala Glu Arg Gly Glu Ala Trp Ala Leu Met
95 100 105 aag gag atc gag gcg gcg ggg gag gcg ctg cag agc gtg cac
gcg gtg 387 Lys Glu Ile Glu Ala Ala Gly Glu Ala Leu Gln Ser Val His
Ala Val 110 115 120 ttt tcg gcg ccc gcc gtc ccc agc ggc acc ggg cag
acg tcg gcg gag 435 Phe Ser Ala Pro Ala Val Pro Ser Gly Thr Gly Gln
Thr Ser Ala Glu 125 130 135 ctg gag gtg cag cgc agg cac tcg ctg gtc
tcg ttt gtg gtg cgc atc 483 Leu Glu Val Gln Arg Arg His Ser Leu Val
Ser Phe Val Val Arg Ile 140 145 150 155 gtg ccc agc ccc gac tgg ttc
gtg ggc gtg gac agc ctg gac ctg tgc 531 Val Pro Ser Pro Asp Trp Phe
Val Gly Val Asp Ser Leu Asp Leu Cys 160 165 170 gac ggg gac cgt tgg
cgg gaa cag gcg gcg ctg gac ctg tac ccc tac 579 Asp Gly Asp Arg Trp
Arg Glu Gln Ala Ala Leu Asp Leu Tyr Pro Tyr 175 180 185 gac gcc ggg
acg gac agc ggc ttc acc ttc tcc tcc ccc aac ttc gcc 627 Asp Ala Gly
Thr Asp Ser Gly Phe Thr Phe Ser Ser Pro Asn Phe Ala 190 195 200 acc
atc ccg cag gac acg gtg acc gag ata acg tcc tcc tct ccc agc 675 Thr
Ile Pro Gln Asp Thr Val Thr Glu Ile Thr Ser Ser Ser Pro Ser 205 210
215 cac ccg gcc aac tcc ttc tac tac ccg cgg ctg aag gcc ctg cct ccc
723 His Pro Ala Asn Ser Phe Tyr Tyr Pro Arg Leu Lys Ala Leu Pro Pro
220 225 230 235 atc gcc agg gtg aca ctg gtg cgg ctg cga cag agc ccc
agg gcc ttc 771 Ile Ala Arg Val Thr Leu Val Arg Leu Arg Gln Ser Pro
Arg Ala Phe 240 245 250 atc cct ccc gcc cca gtc ctg ccc agc agg gac
aat gag att gta gac 819 Ile Pro Pro Ala Pro Val Leu Pro Ser Arg Asp
Asn Glu Ile Val Asp 255 260 265 agc gcc tca gtt cca gaa acg ccg ctg
gac tgc gag gtc tcc ctg tgg 867 Ser Ala Ser Val Pro Glu Thr Pro Leu
Asp Cys Glu Val Ser Leu Trp 270 275 280 tcg tcc tgg gga ctg tgc gga
ggc cac tgt ggg agg ctc ggg acc aag 915 Ser Ser Trp Gly Leu Cys Gly
Gly His Cys Gly Arg Leu Gly Thr Lys 285 290 295 agc agg act cgc tac
gtc cgg gtc cag ccc gcc aac aac ggg agc ccc 963 Ser Arg Thr Arg Tyr
Val Arg Val Gln Pro Ala Asn Asn Gly Ser Pro 300 305 310 315 tgc ccc
gag ctc gaa gaa gag gct gag tgc gtc cct gat aac tgc gtc 1011 Cys
Pro Glu Leu Glu Glu Glu Ala Glu Cys Val Pro Asp Asn Cys Val 320 325
330 taagaccaga gccccgcagc ccctggggcc ccccggagcc atggggtgtc
gggggctcct 1071 gtgcaggctc atgctgcagg cggccgaggg caca 1105 2 331
PRT Homo sapiens 2 Met Glu Asn Pro Ser Pro Ala Ala Ala Leu Gly Lys
Ala Leu Cys Ala 1 5 10 15 Leu Leu Leu Ala Thr Leu Gly Ala Ala Gly
Gln Pro Leu Gly Gly Glu 20 25 30 Ser Ile Cys Ser Ala Arg Ala Leu
Ala Lys Tyr Ser Ile Thr Phe Thr 35 40 45 Gly Lys Trp Ser Gln Thr
Ala Phe Pro Lys Gln Tyr Pro Leu Phe Arg 50 55 60 Pro Pro Ala Gln
Trp Ser Ser Leu Leu Gly Ala Ala His Ser Ser Asp 65 70 75 80 Tyr Ser
Met Trp Arg Lys Asn Gln Tyr Val Ser Asn Gly Leu Arg Asp 85 90 95
Phe Ala Glu Arg Gly Glu Ala Trp Ala Leu Met Lys Glu Ile Glu Ala 100
105 110 Ala Gly Glu Ala Leu Gln Ser Val His Ala Val Phe Ser Ala Pro
Ala 115 120 125 Val Pro Ser Gly Thr Gly Gln Thr Ser Ala Glu Leu Glu
Val Gln Arg 130 135 140 Arg His Ser Leu Val Ser Phe Val Val Arg Ile
Val Pro Ser Pro Asp 145 150 155 160 Trp Phe Val Gly Val Asp Ser Leu
Asp Leu Cys Asp Gly Asp Arg Trp 165 170 175 Arg Glu Gln Ala Ala Leu
Asp Leu Tyr Pro Tyr Asp Ala Gly Thr Asp 180 185 190 Ser Gly Phe Thr
Phe Ser Ser Pro Asn Phe Ala Thr Ile Pro Gln Asp 195 200 205 Thr Val
Thr Glu Ile Thr Ser Ser Ser Pro Ser His Pro Ala Asn Ser 210 215 220
Phe Tyr Tyr Pro Arg Leu Lys Ala Leu Pro Pro Ile Ala Arg Val Thr 225
230 235 240 Leu Val Arg Leu Arg Gln Ser Pro Arg Ala Phe Ile Pro Pro
Ala Pro 245 250 255 Val Leu Pro Ser Arg Asp Asn Glu Ile Val Asp Ser
Ala Ser Val Pro 260 265 270 Glu Thr Pro Leu Asp Cys Glu Val Ser Leu
Trp Ser Ser Trp Gly Leu 275 280 285 Cys Gly Gly His Cys Gly Arg Leu
Gly Thr Lys Ser Arg Thr Arg Tyr 290 295 300 Val Arg Val Gln Pro Ala
Asn Asn Gly Ser Pro Cys Pro Glu Leu Glu 305 310 315 320 Glu Glu Ala
Glu Cys Val Pro Asp Asn Cys Val 325 330 3 36 DNA Artificial
sequence 5' primer containing a BamHI restriction enzyme site
followed by 21 nucleotides of NAD-1 coding sequence. 3 gccatacggg
atccccagcc tcttggggga gagtcc 36 4 35 DNA Artificial sequence 3'
primer containing complementary sequence to an XbaI site followed
by 21 nucleotides of NAF-1 sequence. 4 ggcatacgtc tagattagac
gcagttatca gggac 35 5 41 DNA Artificial sequence 5' primer
containing a BamHI restriction enzyme site followed by 8
nucleotides resembling an efficient signal for initiation of
translation in eukaryotic cells followed by 21 nucleotides of NAF-1
sequence. 5 gccatacggg atccgccatc atggaaaacc ccagcccggc c 41 6 35
DNA Artificial sequence 3' primer containing the cleavage site for
XbaI restriction endonuclease and 21 nucleotides complementary to
the 3' end of the translated sequence of the NAF-1 gene. 6
ggcatacgtc tagattagac gcagttatca gggac 35 7 392 PRT Rattus
norvegicus 7 Pro Thr Gly Thr Gly Cys Val Ile Leu Lys Ala Ser Ile
Val Gln Lys 1 5 10 15 Arg Ile Ile Tyr Phe Gln Asp Glu Gly Ser Leu
Thr Lys Lys Leu Cys 20 25 30 Glu Gln Asp Pro Thr Leu Asp Gly Val
Thr Asp Arg Pro Ile Leu Asp 35 40 45 Cys Cys Ala Cys Gly Thr Ala
Lys Tyr Arg Leu Thr Phe Tyr Gly Asn 50 55 60 Trp Ser Glu Lys Thr
His Pro Lys Asp Tyr Pro Arg Arg Ala Asn His 65 70 75 80 Trp Ser Ala
Ile Ile Gly Gly Ser His Ser Lys Asn Tyr Val Leu Trp 85 90 95 Glu
Tyr Gly Gly Tyr Ala Ser Glu Gly Val Lys Gln Val Ala Glu Leu 100 105
110 Gly Ser Pro Val Lys Met Glu Glu Glu Ile Arg Gln Gln Ser Asp Glu
115 120 125 Val Leu Thr Val Ile Lys Ala Lys Ala Gln Trp Pro Ser Trp
Gln Pro 130 135 140 Val Asn Val Arg Ala Ala Pro Ser Ala Glu Phe Ser
Val Asp Arg Thr 145 150 155 160 Arg His Leu Met Ser Phe Leu Thr Met
Met Gly Pro Ser Pro Asp Trp 165 170 175 Asn Val Gly Leu Ser Ala Glu
Asp Leu Cys Thr Lys Glu Cys Gly Trp 180 185 190 Val Gln Lys Val Val
Gln Asp Leu Ile Pro Trp Asp Ala Gly Thr Asp 195 200 205 Ser Gly Val
Thr Tyr Glu Ser Pro Asn Lys Pro Thr Ile Pro Gln Glu 210 215 220 Lys
Ile Arg Pro Leu Thr Ser Leu Asp His Pro Gln Ser Pro Phe Tyr 225 230
235 240 Asp Pro Glu Gly Gly Ser Ile Thr Gln Val Ala Arg Val Val Ile
Glu 245 250 255 Arg Ile Ala Arg Lys Gly Glu Gln Cys Asn Ile Val Pro
Asp Asn Val 260 265 270 Asp Asp Ile Val Ala Asp Leu Ala Pro Glu Glu
Lys Asp Glu Asp Asp 275 280 285 Thr Pro Glu Thr Cys Ile Tyr Ser Asn
Trp Ser Pro Trp Ser Ala Cys 290 295 300 Ser Ser Ser Thr Cys Glu Lys
Gly Lys Arg Met Arg Gln Arg Met Leu 305 310 315 320 Lys Ala Gln Leu
Asp Leu Ser Val Pro Cys Pro Asp Thr Gln Asp Phe 325 330 335 Gln Pro
Cys Met Gly Pro Gly Cys Ser Asp Glu Asp Gly Ser Thr Cys 340 345 350
Thr Met Ser Glu Trp Ile Thr Trp Ser Pro Cys Ser Val Ser Cys Gly 355
360 365 Met Gly Met Arg Ser Arg Glu Arg Tyr Val Lys Gln Phe Pro Glu
Asp 370 375 380 Gly Ser Val Cys Met Leu Pro Thr 385 390 8 52 PRT
Rattus norvegicus 8 Cys Ile Tyr Ser Asn Trp Ser Pro Trp Ser Ala Cys
Ser Ser Ser Thr 1 5 10 15 Cys Glu Lys Gly Lys Arg Met Arg Gln Arg
Met Leu Lys Ala Gln Leu 20 25 30 Asp Leu Ser Val Pro Cys Pro Asp
Thr Gln Asp Phe Gln Pro Cys Met 35 40 45 Gly Pro Gly Cys 50 9 53
PRT Rattus norvegicus 9 Cys Thr Met Ser Glu Trp Ile Thr Trp Ser Pro
Cys Ser Val Ser Cys 1 5 10 15 Gly Met Gly Met Arg Ser Arg Glu Arg
Tyr Val Lys Gln Phe Pro Glu 20 25 30 Asp Gly Ser Val Cys Met Leu
Pro Thr Glu Glu Thr Glu Lys Cys Thr 35 40 45 Val Asn Glu Glu Cys 50
10 52 PRT Rattus norvegicus 10 Cys Leu Val Thr Glu Trp Gly Glu Trp
Asp Asp Cys Ser Ala Thr Cys 1 5 10 15 Gly Met Gly Met Lys Lys Arg
His Arg Met Val Lys Met Ser Pro Ala 20 25 30 Asp Gly Ser Met Cys
Lys Ala Glu Thr Ser Gln Ala Glu Lys Cys Met 35 40 45 Met Pro Glu
Cys 50 11 51 PRT Rattus norvegicus 11 Cys Leu Leu Ser Pro Trp Ser
Glu Trp Ser Asp Cys Ser Val Thr Cys 1 5 10 15 Gly Lys Gly Met Arg
Thr Arg Gln Arg Met Leu Lys Ser Leu Ala Glu 20 25 30 Leu Gly Asp
Cys Asn Glu Asp Leu Glu Gln Ala Glu Lys Cys Met Leu 35 40 45 Pro
Glu Cys 50 12 52 PRT Rattus norvegicus 12 Cys Glu Leu Ser Glu Trp
Ser Gln Trp Ser Glu Cys Asn Lys Ser Cys 1 5 10 15 Gly Lys Gly His
Met Ile Arg Thr Arg Thr Ile Gln Met Glu Pro Gln 20 25 30 Phe Gly
Gly Ala Pro Cys Pro Glu Thr Val Gln Arg Lys Lys Cys Arg 35 40 45
Ala Arg Lys Cys 50 13 53 PRT Rattus norvegicus 13 Cys Arg Met Arg
Pro Trp Thr Ala Trp Ser Glu Cys Thr Lys Leu Cys 1 5 10 15 Gly Gly
Gly Ile Gln Glu Arg Tyr Met Thr Val Lys Lys Arg Phe Lys 20 25 30
Ser Ser Gln Phe Thr Ser Cys Lys Asp Lys Lys Glu Ile Arg Ala Cys 35
40 45 Asn Val His Pro Cys 50 14 50 PRT Homo sapiens 14 Cys Leu Val
Ser Glu Trp Ser Glu Trp Ser Asp Cys Ser Thr Cys Gly 1 5 10 15 Lys
Gly Met Arg Ser Arg Thr Arg Met Val Lys Met Ser Pro Ala Asp 20 25
30 Gly Ser Pro Cys Pro Asp Thr Glu Glu Ala Glu Lys Cys Met Val Pro
35 40 45 Glu Cys 50 15 506 DNA Homo sapiens misc_feature (11)..(11)
n is equal to a, t, c, or g 15 gaattcggca naggnnaaac cccagcccgg
ctgccgccct gggcaaggcc tnctgcgctc 60 tcctcctggc cactctcggc
gccggcacca gcctcttggg ggagagtcca tctnttccgc 120 cagagccccg
gccaaataca gcatcacctt cacgggcaag tggagccaga cggccttccc 180
caagcagtac cccctgttcc gcccccctgc gcatggtntt cgctgctggg ggccgcgcat
240 agctccgact acagcatgtg gaggaagaac cagtacgtca taaacgggct
gcgcgacttt 300 ncggagcggc gaggcctngg ncgttgatga aggagatccg
ggnggcgggg gaggcgtnca 360 anaggtgnca agagttnttt tcggggcccg
gttccccaan ggnaacnggn aaacgttggg 420 ggntttnnag tttnaagaag
naattnttgg tttttttttg ggtgggattt tnccaacccn 480 attgtttntg
ggntggaaaa ttngac 506 16 316 DNA Homo sapiens misc_feature (5)..(5)
n is equal to a, t, c, or g 16 ggcanngcca gtacgtcata acgggctgcg
cgactttgcg gangcggcga ggcctgggcg 60 ctgatgaagg agatcaaggc
ggcgggggag gcgctgcaga ggtgcacgag gtgttttcgg 120 cgcccggtnn
cccagcgnca ccnggcagac gtcggcgaac tggnaggtgc agcgcaggca 180
ctcgctggtc tcgtttgtgg tgcgcatcgt gcccagcccc gactggttcg tgggcgtgga
240 cagcctggga cctgtganaa cggggacctt tngcgngnaa caggcgncgt
tggacctgta 300 nccctacgac gncggg 316 17 316 DNA Homo sapiens
misc_feature (5)..(5) n is equal to a, t, c, or g 17 ggcanngcca
gtacgtcata acgggctgcg cgactttgcg gangcggcga ggcctgggcg 60
ctgatgaagg agatcaaggc ggcgggggag gcgctgcaga ggtgcacgag gtgttttcgg
120 cgcccggtnn cccagcgnca ccnggcagac gtcggcgaac tggnaggtgc
agcgcaggca 180 ctcgctggtc tcgtttgtgg tgcgcatcgt gcccagcccc
gactggttcg tgggcgtgga 240 cagcctggga cctgtganaa cggggacctt
tngcgngnaa caggcgncgt tggacctgta 300 nccctacgac gncggg 316 18 53
PRT Homo sapiens 18 Cys Glu Val Ser Leu Trp Ser Ser Trp Gly Leu Cys
Gly Gly His Cys 1 5 10 15 Gly Arg Leu Gly Thr Lys Ser Arg Thr Arg
Tyr Val Arg Val Gln Pro 20 25 30 Ala Asn Asn Gly Ser Pro Cys Pro
Glu Leu Glu Glu Glu Ala Glu Cys 35 40 45 Val Pro Asp Asn Cys 50
* * * * *