U.S. patent application number 10/417924 was filed with the patent office on 2003-11-20 for biologically active eph family ligands.
Invention is credited to Davis, Samuel, Gale, Nicholas W., Yancopoulos, George D..
Application Number | 20030215918 10/417924 |
Document ID | / |
Family ID | 21723735 |
Filed Date | 2003-11-20 |
United States Patent
Application |
20030215918 |
Kind Code |
A1 |
Davis, Samuel ; et
al. |
November 20, 2003 |
Biologically active Eph family ligands
Abstract
A novel ligand (Efl-6) that binds the Elk subfamily of Eph
receptors is identified, and methods for making the soluble Elf-6
ligand in biologically active form is described. A cDNA clone
encoding this novel protein enables production of the recombinant
protein, which is useful to support neuronal and other eph
receptor-bearing cell populations.
Inventors: |
Davis, Samuel; (New York,
NY) ; Gale, Nicholas W.; (Yorktown Heights, NY)
; Yancopoulos, George D.; (Yorktown Heights, NY) |
Correspondence
Address: |
REGENERON PHARMACEUTICALS, INC
777 OLD SAW MILL RIVER ROAD
TARRYTOWN
NY
10591
US
|
Family ID: |
21723735 |
Appl. No.: |
10/417924 |
Filed: |
April 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10417924 |
Apr 17, 2003 |
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09051994 |
Apr 24, 1998 |
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6602683 |
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09051994 |
Apr 24, 1998 |
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PCT/US96/17201 |
Oct 25, 1996 |
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60007015 |
Oct 25, 1995 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/7.5; 514/8.3; 530/350; 536/23.5 |
Current CPC
Class: |
A61P 25/00 20180101;
C07K 14/4702 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 514/12; 530/350; 536/23.5 |
International
Class: |
A61K 038/17; C07K
014/705; C12P 021/02; C12N 005/06; C07H 021/04 |
Claims
1. An isolated and purified nucleic acid molecule encoding Efl-6
protein wherein the sequence of said nucleic acid is selected from
the group consisting of: (a) the sequence of the DNA encoding
mature Efl-6 protein contained in the plasmid pBluescriptSK.sup.14
Efl-6 as deposited with the American Type Culture Collection on
Oct. 19, 1995 and designated as 97319; (b) the sequence of the DNA
encoding mature Efl-6 protein as set forth in FIG. 1; (c) DNA
sequences that hybridize under moderately stringent conditions to
the DNA of (a) or (b) and which encode a protein that binds a
receptor belonging to the Elk subfamily of Eph receptors; and (d)
DNA sequences that are degenerate as a result of the genetic code
to a DNA sequence of (a), (b), or (c) and which encode an Efl-6
protein that binds a member of the Elk subclass of Eph
receptors.
2. Isolated and purified mature Efl-6 protein having an amino acid
sequence as set forth in FIG. 1.
3. An isolated nucleic acid encoding the extracellular domain of
Efl-6 (sEfl-6) having a sequence selected from the following: (a)
the sequence set forth from about nucleotide 274 to about
nucleotide 873 of FIG. 1; and (b) a sequence which encodes the
extracellular domain of Efl-6 as set forth in FIG. 1.
4. Purified sEfl-6 encoded by the nucleotide sequence of claim
3.
5. (sEfl-6)n comprising the sEfl-6 protein according to claim 4,
wherein n is 2 or greater.
6. Efl-6 ligandbody comprising soluble Efl-6 protein according to
claim 4 and the Fc portion of IgG.
7. A vector which comprises a nucleic acid molecule of claim 1.
8. A vector according to claim 7 wherein the nucleic acid molecule
is operatively linked to an expression control sequence capable of
directing its expression in a host cell.
9. A host cell containing a vector according to claim 8.
10. A vector which comprises a nucleic acid molecule of claim
3.
11. A vector according to claim 10 wherein the nucleic acid
molecule is operatively linked to an expression control sequence
capable of directing its expression in a host cell.
12. A host cell containing a vector according to claim 11.
13. A method of producing Efl-6 ligand which comprises growing
cells of a host according to claim 8 under conditions permitting
production of the ligand, and recovering the ligand so
produced.
14. A method of producing Efl-6 soluble ligand which comprises
growing cells of a host according to claim 11 under conditions
permitting production of the ligand, and recovering the ligand so
produced.
15. An antibody which specifically binds the ligand of claim 2 or
4.
16. An antibody according to claim 15 which is a monoclonal
antibody.
Description
INTRODUCTION
[0001] The present invention provides for a novel ligand that binds
proteins belonging to the Eph subfamily of receptorlike protein
tyrosine kinases, such as the Elk receptor and methods for making
soluble forms of this ligand that are biologically active.
BACKGROUND OF THE INVENTION
[0002] The ability of polypeptide ligands to bind cells and thereby
elicit a phenotypic response such as cell growth, survival or
differentiation is often mediated through transmembrane tyrosine
kinases. The extracellular portion of each receptor tyrosine kinase
(RTK) is generally the most distinctive portion of the molecule, as
it provides the protein with its ligand-recognizing characteristic.
Binding of a ligand to the extracellular domain results in signal
transduction via an intracellular tyrosine kinase catalytic domain
which transmits a biological signal to intracellular target
proteins. The particular array of sequence motifs of this
cytoplasmic, catalytic domain determines its access to potential
kinase substrates (Mohammadi, et al., 1990, Mol. Cell. Biol., 11:
5068-5078; Fantl, et al., 1992, Cell, 69:413-413).
[0003] RTKs appear to undergo dimerization or some related
conformational change following ligand binding (Schlessinger, J.,
1988, Trend Biochem. Sci. 13:443-447; Ullrich and Schlessinger,
1990, Cell, 61:203-212; Schlessinger and Ullrich, 1992, Neuron
9:383-391); molecular interactions between dimerizing cytoplasmic
domains lead to activation of kinase function. In some instances,
such as the growth factor platelet derived growth factor (PDGF),
the ligand is a dimer that binds two receptor molecules (Hart, et
al. 1988, Science, 240: 1529-1531; Heldin, 1989, J. Biol. Chem.
264:8905-8912) while, for example, in the case of EGF, the ligand
is a monomer (Weber, et al., 1984, J. Biol. Chem.,
259:14631-14636).
[0004] The tissue distribution of a particular tyrosine kinase
receptor within higher organisms provides relevant data as to the
biological function of the receptor. The tyrosine kinase receptors
for some growth and differentiation factors, such as fibroblast
growth factor (FGF) are widely expressed and therefore appear to
play some general role in tissue growth and maintenance. Members of
the Trk RTK family (Glass & Yancopoulos, 1993, Trends in Cell
Biol, 3:262-268) of receptors are more generally limited to cells
of the nervous system, and the Nerve Growth Factor family
consisting of NGF, BDNF, NT-3 and NT-4/5 (known as the
neurotrophins) which bind these receptors promote the
differentiation of diverse groups of neurons in the brain and
periphery (Lindsay, R. M, 1993, in Neurotrophic Factors, S. E.
Loughlin & J. H. Fallon, eds., pp. 257-284 (San Diego, Calif.:
Academic Press). The localization of one such Trk family receptor,
trkB, in tissue provided some insight into the potential biological
role of this receptor, as well as the ligands that bind this
receptor (referred to herein as cognates). Thus, for example, in
adult mice, trkB was found to be preferentially expressed in brain
tissue, although significant levels of trkB mRNAs were also
observed in lung, muscle, and ovaries. Further, trkB transcripts
were detected in mid and late gestation embryos. In situ
hybridization analysis of 14 and 18 day old mouse embryos indicated
that trkB transcripts were localized in the central and peripheral
nervous systems, including brain, spinal cord, spinal and cranial
ganglia, paravertebral trunk of the sympathetic nervous system and
various innervation pathways, suggesting that the trkB gene product
may be a receptor involved in neurogenesis and early neural
development as well as play a role in the adult nervous system.
[0005] The cellular environment in which an RTK is expressed may
influence the biological response exhibited upon binding of a
ligand to the receptor. Thus, for example, when a neuronal cell
expressing a Trk receptor is exposed to a neurotrophin which binds
that receptor, neuronal survival and differentiation results. When
the same receptor is expressed by a fibroblast, exposure to the
neurotrophin results in proliferation of the fibroblast (Glass, et
al., 1991, Cell 66:405-413). Thus, it appears that the
extracellular domain provides the determining factor as to the
ligand specificity, and once signal transduction is initiated the
cellular environment will determine the phenotypic outcome of that
signal transduction.
[0006] A number of RTK families have been identified based on
sequence homologies in their intracellular domain. The receptor and
signal transduction pathways utilized by NGF involves the product
of the trk proto-oncogene (Kaplan et al., 1991, Nature 350:156-160;
Klein et al., 1991, Cell 65:189-197). Klein et al. (1989, EMBO J.
8:3701-3709) reported the isolation of trkB, which encodes a second
member of the tyrosine protein kinase family of receptors found to
be highly related to the human trk protooncogene. TrkB binds and
mediates the functional responses to BDNF, NT-4, and, to a lesser
extent, NT-3 (Squinto, et al., 1991, Cell 65:885-903; Ip, et al.,
1992, Proc. Natl. Acad. Sci. U.S.A. 89:3060-3064; Klein, et al.,
1992, Neuron, 8:947-956). At the amino acid level, the products of
trk and trkB were found to share 57 percent homology in their
extracellular regions, including 9 of the 11 cysteines present in
trk. This homology was found to increase to 88 percent within their
respective tyrosine kinase catalytic domains. The Trk gene family
has now been expanded to include the trkC locus, with NT-3 having
been identified as the preferred ligand for trkC (Lamballe, et al.,
1991, Cell 66: 967-979; Valenzuela, et al. 1993, Neuron 10:963-1 5
974).
[0007] The Eph-related transmembrane tyrosine kinases comprise the
largest known family of receptor-like tyrosine kinases, with many
members displaying specific expression in the developing and adult
nervous system. Two novel members of the Eph RTK family, termed Ehk
(eph homology kinase)-1 and -2 were identified using a polymerase
chain reaction (PCR)-based screen of genes expressed in brain
(Maisonpierre, et al. 1993, Oncogene 8:3277-388). These genes
appear to be expressed exclusively in the nervous system, with
Ehk-1 expression beginning early in neural development. Recently, a
new member of this group of related receptors, Ehk-3 has been
cloned (Valenzuela, et al. 1995, Oncogene 10:1573-1580).
[0008] The elk gene encodes a receptorlike protein-tyrosine kinase
that also belongs to the eph subfamily, and which is expressed
almost exclusively in the brain (and at lower levels in the testes)
(Letwin, et al. 1988; Oncogene 3:621-678; Lhotak, et al., 1991 Mol.
Cell. Biol. 11: 2496-2502). Based on its expression profile, the
Elk receptor and its cognate ligand are expected to play a role in
cell to cell interactions in the nervous system. Other members of
the Eph family of receptors that fall within the same subclass as
Elk include the Nuk/Cek5, Hek2/Sek4 and Htk receptors (Brambilia
and Klein, 1995, Mol. Cell. Neurosci., 6:487-495, Gale, et al.,
1996, Neuron 17:9-19).
[0009] Unlike the Ehks and Elk receptors, the closely related Eck
receptor appears to function in a more pleiotropic manner; it has
been identified in neural, epithelial and skeletal tissues and it
appears to be involved in the gastrulation, craniofacial, and limb
bud sites of pattern formation in the mouse embryo (Ganju, et al.
1994, Oncogene 9:1613-1624).
[0010] The identification of a large number of receptor tyrosine
kinases has far exceeded the identification of their cognate
ligands. At best, determination of the tissues in which such
receptors are expressed provides insight into the regulation of the
growth, proliferation and regeneration of cells in target tissues.
Because RTKs appear to mediate a number of important functions
during development, their cognate ligands will inevitably play a
crucial role in development.
[0011] Although a number of schemes have been devised for the
identification of cognate ligands for the many orphan receptors
that have been identified, very few such ligands have been
identified, and the ligands that have been identified to date
appear to have no activity other than the ability to bind their
cognate receptor. For example, International Publication Number
WO/94/11020 published on May 26, 1994 describes ligands that bind
to the Eck receptor. In particular the ligand EBP (also known as
B61) is described. However, although binding of B61 to the Eck
receptor is disclosed, no biological activity is described.
Similarly, despite the description in PCT Publication Number
WO94/11384 (published May 26, 1994) of a ligand that binds the Elk
receptor, no biological activity was observed, regardless of
whether the ligand was presented as membrane bound or in the form
of an Fc dimer of the soluble ligand. With respect to the Elk
receptor, however, chimeric EGFR-Elk receptors (having the
extracellular domain of the EGFR fused to the Elk cytoplasmic
domain) have been used to demonstrate the functional integrity (as
measured by EGF-stimulated autophosphorylation) of the enzymatic
domain of this receptor. (Lhotak and Pawson, 1993, Mol. Cell. Biol.
13:7071-7079).
SUMMARY OF THE INVENTION
[0012] The present invention provides for a novel polypeptide
ligand, designated as Efl-6, that binds to the Elk, Nuk/Cek5,
Hek2/Sek4, Htk, and Sek1 receptors on cells. More importantly, the
invention provides a means of making biologically active, soluble
forms of this ligand, which are useful in promoting a differential
function and/or influencing the phenotype, such as growth and/or
proliferation, of receptor bearing cells. The invention also
provides for nucleic acids encoding such polypeptide ligands, and
both prokaryotic and eukaryotic expression systems for producing
such proteins. The invention also provides for antibodies to these
ligands.
[0013] According to the invention, soluble forms of the ligands
described herein may be used to promote biological responses in
Elk, Nuk/Cek5, Hek2/Sek4, Htk, and Sek1 receptor-expressing cells.
In particular, a general method is described herein which produces
"clustering" of ligands for eph-related receptors, which functions
to make otherwise inactive soluble ligands biologically active, or
which enhances the biological activity of ligands that, absent such
clustering, would have only low levels of biological activity.
[0014] The ligands described herein also have diagnostic utilities.
In particular embodiments of the invention, methods of detecting
aberrancies in their function or expression may be used in the
diagnosis of neurological or other disorders.
[0015] In other embodiments, manipulation of the interaction
between the ligands and their cognate receptor may be used in assay
systems designed to identify both agonists and antagonists of Eph
receptor ligands. Such agonists and antagonists may be developed
for use in the eventual treatment of neurological or other
disorders.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1. Nucleotide and encoded protein sequence of Efl-6.
The putative signal sequence is encoded by about nucleotide 202 to
about nucleotide 273. The coding region of the mature protein
begins at about nucleotide 274 and ends at about nucleotide 1224.
The deposited clone has an A at position 698. This change created
an amino acid change from Q (Gln) to R (Arg). The coding region for
the putative transmembrane domain is shown underlined. The amino
acid sequence of the encoded extracellular domain, which is encoded
by about nucleotide 274 to about nucleotide 873, is shown in bold
letters.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention provides for a novel polypeptide
ligand that binds to the Elk receptor. The novel polypeptide ligand
of the present invention is also able to bind other members of the
Elk subclass of Eph receptors, including Nuk/Cek5, Hek2/Sek4 and
Htk, as well as the only receptor known to "cross subclasses",
known as Sek1 (Brambilla and Klein, 1995, Mol. Cell. Neurosci.,
6:487-495, Gale, et al., 1996, Neuron 17:9-19). Accordingly, as
used herein, the "Elk" receptor refers to Elk, as well as the above
receptors known to bind the Elk ligands.
[0018] The invention further provides a means of making
biologically active, soluble forms of the Efl-6 ligand, which are
useful in promoting a differential function and/or influencing the
phenotype, such as growth and/or proliferation, of receptor bearing
cells. The invention also provides for nucleic acids encoding such
a polypeptide ligand, and both prokaryotic and eukaryotic
expression systems for producing this protein. The invention also
provides for antibodies to this ligand.
[0019] The novel ligand described herein is designated as Efl (Eph
transmembrane tyrosine kinase family ligands)-6. A deposit
designated as pbluescript SK-encoding Efl-6 was made with the
American Type Culture Collection on Oct. 19, 1995 and has received
accession number 97319.
[0020] According to the invention, soluble forms of the Elk ligand
(referred to herein as Efl-6) may be used to promote biological
responses in Elk receptor-expressing cells. In particular, a
general method is described herein which produces "clustering" of
Efl-6 ligand which functions to make otherwise inactive soluble
ligand biologically active, or which enhances the biological
activity of the ligand which, absent such clustering, would have
only low levels of biological activity.
[0021] The Efl-6 ligand described herein may also have diagnostic
utilities. In particular embodiments of the invention, methods of
detecting aberrancies in its function or expression may be used in
the diagnosis of neurological or other disorders. In other
embodiments, manipulation of the interaction between the ligand and
its cognate receptor may be used in the treatment of neurological
or other disorders.
[0022] When used herein, Efl-6 includes functionally equivalent
molecules in which amino acid residues are substituted for residues
within the sequence resulting in a silent change. For example, one
or more amino acid residues within the sequence can be substituted
by another amino acid of a similar polarity which acts as a
functional equivalent, resulting in a silent alteration.
Substitutes for an amino acid within the sequence may be selected
from other members of the class to which the amino acid belongs.
For example, the nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan and methionine. The polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine. The positively charged (basic) amino acids include
arginine, lysine and histidine. The negatively charged (acidic)
amino acids include aspartic acid and glutamic acid. Also included
within the scope of the invention are proteins or fragments or
derivatives thereof which exhibit the same or similar biological
activity and derivatives which are differentially modified during
or after translation, e.g., by glycosylation, proteolytic cleavage,
linkage to an antibody molecule or other cellular ligand, etc.
[0023] Cells that express Efl-6 may do so naturally or may be
genetically engineered to produce this ligand, as described supra,
by transfection, transduction, electroporation, microinjection, via
a transgenic animal, etc. of nucleic acid encoding Efl-6 described
herein in a suitable expression vector. A vector containing the
cDNA encoding for EFl-6 deposited with the American Type Culture
Collection under the terms of the Budapest Treaty on Oct. 19, 1995
as pBluescriptSK-Efl-6 has been given the ATCC designation
97319.
[0024] The present invention encompasses the DNA sequence contained
in the above deposited plasmid, as well as DNA and RNA sequences
that hybridize to the Efl-6 encoding sequence contained therein,
under conditions of moderate stringency, as defined in, for
example, Sambrook, et al. Molecular Cloning: A Laboratory Manual, 2
ed. Vol. 1, pp. 101-104, Cold Spring Harbor Laboratory Press
(1989). Thus, nucleic acids contemplated by the invention include
the sequence as contained in the deposit and as set forth in FIG.
1, sequences of nucleic acids that hybridize to such sequence and
which bind the Elk receptor, and nucleic acid sequences which are
degenerate of the above sequences as a result of the genetic code,
but which encode ligand(s) that bind the Elk receptor.
[0025] In addition, the present invention contemplates use of the
ligands described herein in soluble forms, truncated forms, and
tagged forms. This includes monomeric forms of the ligand which may
bind to the receptor and function as an antagonist.
[0026] Any of the methods known to one skilled in the art for the
insertion of DNA fragments into a vector may be used to construct
expression vectors encoding Efl-6 using appropriate
transcriptional/translational control signals and the protein
coding sequences. These methods may include in vitro recombinant
DNA and synthetic techniques and in vivo recombinations (genetic
recombination). Expression of nucleic acid sequence encoding the
Efl-6 or peptide fragments thereof may be regulated by a second
nucleic acid sequence so that the protein or peptide is expressed
in a host transformed with the recombinant DNA molecule. For
example, expression of the Efl-6 described herein may be controlled
by any promoter/enhancer element known in the art. Promoters which
may. be used to control expression of the ligands include, but are
not limited to the long terminal repeat as described in Squinto et
al., (1991, Cell 65:1-20); the SV40 early promoter region (Bernoist
and Chambon, 1981, Nature 290:304-310), the CMV promoter, the
M-MuLV 5' terminal repeat the promoter contained in the 3' long
terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell
22:787-797), the herpes thymidine kinase promoter (Wagner et al.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:144-1445), the regulatory
sequences of the metallothioein gene (Brinster et al., 1982, Nature
296:39-42); prokaryotic expression vectors such as the
.beta.-lactamase promoter (Villa-Kamaroff, et al., 1978, Proc.
Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer,
et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), see also
"Useful proteins from recombinant bacteria" in Scientific American,
1980, 242:74-94; promoter elements from yeast or other fungi such
as the Gal 4 promoter, the ADH (alcohol dehydrogenase) promoter,
PGK (phosphoglycerol kinase) promoter, alkaline phosphatase
promoter, and the following animal transcriptional control regions,
which exhibit tissue specificity and have been utilized in
transgenic animals: elastase I gene control region which is active
in pancreatic acinar cells (Swift, et al., 1984, Cell 38:639-646;
Ornitz, et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene
control region which is active in pancreatic beta cells (Hanahan,
1985, Nature 315:115-122), immunoglobulin gene control region which
is active in lymphoid cells (Grosschedl, et al., 1984, Cell
38:647-658; Adames, et al., 1985, Nature 318:533-538; Alexander et
al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus
control region which is active in testicular, breast, lymphoid and
mast cells (Leder, et al., 1986, Cell 45:485-495), albumin gene
control region which is active in liver (Pinkert, et al., 1987,
Genes and Devel. 1:268-276), alpha-fetoprotein gene control region
which is active in liver (Krumlauf, et al., 1985, Mol. Cell. Biol.
5:1639-1648; Hammer et al., 1987, Science 235:53-58); alpha
1-antitrypsin gene control region which is active in the liver
(Kelsey, et al, 1987, Genes and Devel. 1:161-171), beta-globin gene
control region which is active in myeloid cells (Mogram, et al.,
1985, Nature 315:338-340; Kollias, et al., 1986, Cell 46:89-94);
myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain (Readhead, et al., 1987, Cell
48:703-712); myosin light chain-2 gene control region which is
active in skeletal muscle (Shani, 1985, Nature 314:283-286), and
gonadotropic releasing hormone gene control region which is active
in the hypothalamus (Mason et al., 1986, Science
234:1372-1378).
[0027] Thus, according to the invention, expression vectors capable
of being replicated in a bacterial or eukaryotic host comprising
Efl-6 encoding nucleic acid as described herein, are used to
transfect the host and thereby direct expression of such nucleic
acid to produce the Efl-6 proteins, which may then be recovered in
biologically active form. As used herein, a biologically active
form includes a form capable of binding to the relevant receptor,
such as Elk, and causing a differentiated function and/or
influencing the phenotype of the cell expressing the receptor. Such
biologically active forms would, for example, induce
phosphorylation of the tyrosine kinase domain of the Elk receptor,
or stimulation of synthesis of cellular DNA. Alternatively,
biologically active Elf-6 ligand includes monomeric forms that bind
the receptor and act as antagonists.
[0028] Expression vectors containing the gene inserts can be
identified by three general approaches: (a) DNA-DNA hybridization,
(b) presence or absence of "marker" gene functions, and (c)
expression of inserted sequences. In the first approach, the
presence of a foreign gene inserted in an expression vector can be
detected by DNA-DNA hybridization using probes comprising sequences
that are homologous to an inserted efl-6 gene. In the second
approach, the recombinant vector/host system can be identified and
selected based upon the presence or absence of certain "marker"
gene functions (e.g., thymidine kinase activity, resistance to
antibiotics, transformation phenotype, occlusion body formation in
baculovirus, etc.) caused by the insertion of foreign genes in the
vector. For example, if the efl-6 gene is inserted within the
marker gene sequence of the vector, recombinants containing the
insert can be identified by the absence of the marker gene
function. In the third approach, recombinant expression vectors can
be identified by assaying the foreign gene product expressed by the
recombinant. Such assays can be based, for example, on the physical
or functional properties of the efl-6 gene product, for example, by
binding of the ligand to the Elk receptor or portion thereof which
may be tagged with, for example, a detectable antibody or portion
thereof or binding to antibodies produced against the Efl-6 protein
or a portion thereof.
[0029] Efl-6 appears to be a conventional transmembrane protein
with a cytoplasmic domain. The transmembrane domain is shown
underlined in FIG. 1. Accordingly, the soluble or extracellular
domain of the ligand (sEfl-6) is encoded by the nucleotide sequence
from about nucleotide 274 to about nucleotide 873.
[0030] The ligands described herein may be produced as membrane
bound forms in animal cell expression systems or may be expressed
in soluble form. Soluble forms of the ligands may be expressed
using methods known to those in the art. A commonly used strategy
involves use of oligonucleotide primers, one of which spans the
N-terminus of the protein, the other of which spans the region just
upstream to a hydrophobic segment of the protein, which represents
either the GPI-linkage recognition domain or a transmembrane domain
of the protein. The oligonucleotide spanning the C-terminus region
is modified so as to contain a stop codon prior to the hydrophobic
domain. The two oligonucleotides are used to amplify a modified
version of the gene encoding a protein that is secreted instead of
membrane bound. Alternatively, a convenient restriction site in the
vector can be used to insert an altered sequence that removes the
GPI-linkage recognition domain or transmembrane domain, thus
resulting in a vector capable of expressing a secreted form of the
protein. The soluble protein so produced would include the region
of the protein from the N-terminus to the region preceding the
hydrophobic GPI recognition domain or transmembrane domain.
[0031] Applicants have discovered that although the soluble ligands
produced according to the invention bind to the receptors in the
eph subfamily, such soluble ligands often have little or no
biological activity. Such soluble ligands are activated, according
to the present invention, by ligand "clustering". "Clustering" as
used herein refers to any method known to one skilled in the art
for creating multimers of the soluble portions of ligands described
herein.
[0032] In one embodiment, a "clustered" efl-6 is a dimer, made for
example, according to the present invention utilizing the Fc domain
of IgG (Aruffo, et al., 1991, Cell 67:35-44), which results in the
expression of the soluble ligand as a disulfide-linked homodimer.
In another embodiment, secreted forms of the ligand are constructed
with epitope tags at their C-termini; anti-tag antibodies are then
used to aggregate the ligands.
[0033] In addition, the invention contemplates other "engineered"
ligand molecules that exist as or form multimers. For example,
dimers of the extracellular domains may be engineered using leucine
zippers. The leucine zipper domains of the human transcription
factors c-jun and c-fos have been shown to form stable heterodimers
[Busch and Sassone-Corsi, Trends Genetics 6: 36-40 (1990); Gentz,
et al., Science 243: 1695-1699 (1989)] with a 1:1 stoichiometry.
Although jun-jun homodimers have. also been shown to form, they are
about 1000-fold less stable than jun-fos heterodimers. Fos-fos
homodimers have not been detected. The leucine zipper domain of
either c-jun or c-fos are fused in frame at the C-terminus of the
soluble or extracellular domains of the above mentioned ligands by
genetically engineering chimeric genes. The fusions may be direct
or they may employ a flexible linker domain, such as the hinge
region of human IgG, or polypeptide linkers consisting of small
amino acids such as glycine, serine, threonine or alanine, at
various lengths and combinations. Additionally, the chimeric
proteins may be tagged by His-His-His-His-His-His (His6), to allow
rapid purification by metal-chelate chromatography, and/or by
epitopes to which antibodies are available, to allow for detection
on western blots, immunoprecipitation, or activity
depletion/blocking in bioassays.
[0034] Alternatively, multimers may be made by genetically
engineering and expressing molecules that consist of the soluble or
extracellular portion of the ligand followed by the Fc-domain of
hIgG, followed by either the c-jun or the c-fos leucine zippers
described above [Kostelny, et al., J. Immunol. 148: 1547-1553
(1992)]. Since these leucine zippers form predominately
heterodimers, they may be used to drive formation of the
heterodimers where desired. As for the chimeric proteins described
using leucine zippers, these may also be tagged with metal chelates
or an epitope. This tagged domain can be used for rapid
purification by metal-chelate chromatography, and/or by antibodies,
to allow for detection on western blots, immunoprecipitation, or
activity. depletion/blocking in bioassays.
[0035] In another embodiment of the invention, multimeric soluble
ligands are prepared by expression as chimeric molecules utilizing
flexible linker loops. A DNA construct encoding the chimeric
protein is designed such that it expresses two or more soluble or
extracellular domains fused together in tandem ("head to head") by
a flexible loop. This loop may be entirely artificial (e.g.
polyglycine repeats interrupted by serine or threonine at a certain
interval) or "borrowed" from naturally occurring proteins (e.g. the
hinge region of hIgG). Molecules may be engineered in which the
length and composition of the loop is varied, to allow for
selection of molecules with desired characteristics. Although not
wishing to be bound by theory, applicants believe that membrane
attachment of the ligands facilitates ligand clustering, which in
turn promotes receptor multimerization and activation. Thus,
according to the invention, biological activity of the soluble
ligand is achieved by mimicking, in solution, membrane associated
ligand clustering. Thus, a biologically active, clustered soluble
eph family ligand comprises (soluble Efl).sub.n, wherein the
soluble efl is the extracellular domain of a ligand that binds an
eph family receptor and n is 2 or greater. As described herein,
Efl-6 is made biologically active according to the process of the
invention.
[0036] In each case, one skilled in the art will recognize that the
success of clustering will require analysis of the biological
activity utilizing bioassays such as those described herein. For
example, receptor phosphorylation induced by stimulating receptor
expressing reporter cells with COS cells overexpressing membrane
forms of the ligands, soluble forms of the ligands and clustered
ligands may be compared.
[0037] Although in some instances dimerization of the ligand is
sufficient to induce biological activity, in certain instances, the
methods described herein are used to determine the sufficiency of a
particular clustering technique. Often dimerization of a soluble
ligand utilizing Fc appears to be insufficient for achieving a
biological response, yet further clustering of the ligand according
to the invention using anti-Fc antibodies may result in a
substantial increase in biological activity.
[0038] Cells of the present invention may transiently or,
preferably, constitutively and permanently express Efl-6 in native
form, or in soluble form as tagged Efl-6 or clustered Efl-6 as
described herein.
[0039] The recombinant factor may be purified by any technique
which allows for the subsequent formation of a stable, biologically
active protein. For example, and not by way of limitation, the
factor may be recovered from cells either as a soluble protein or
as inclusion bodies, from which it may be extracted quantitatively
by 8M guanidinium hydrochloride and dialysis. In order to further
purify the factor, conventional ion exchange chromatography,
hydrophobic interaction chromatography, reverse phase
chromatography or gel filtration may be used.
[0040] In additional embodiments of the invention, recombinant
efl-6 may be used to inactivate or "knock out" the endogenous gene
by homologous recombination, and thereby create an Efl-6 protein
deficient cell, tissue, or animal. For example, and not by way of
limitation, recombinant efl may be engineered to contain an
insertional mutation, for example the neo gene, which would
inactivate the native efl-6 gene. Such a construct, under the
control of a suitable promoter, may be introduced into a cell, such
as an embryonic stem cell, by a technique such as transfection,
transduction, injection, etc. Cells containing the construct may
then be selected by G418 resistance. Cells which lack an intact
efl-6 may then be identified, e.g. by Southern blotting or Northern
blotting or assay of expression. Cells lacking an intact efl-6 may
then be fused to early embryo cells to generate transgenic animals
deficient in such ligand. A comparison of such an animal with an
animal expressing endogenous Efl-6 would aid in the elucidation of
the role of the ligands in development and maintenance. Such an
animal may be used to define specific neuronal populations, or any
other in vivo processes, normally dependent upon the ligand.
[0041] The present invention also provides for antibodies to the
Efl-6 described herein which are useful for detection of the ligand
in, for example, diagnostic applications. Antibodies to the ligand
may also be useful for achieving clustering according to the
invention. In instances where endogenous ligand exists, the
antibody itself may act as the therapeutic by activating existing
ligand.
[0042] For preparation of monoclonal antibodies directed toward
Efl-6, any technique which provides for the production of antibody
molecules by continuous cell lines in culture may be used. For
example, the hybridoma technique originally developed by Kohler and
Milstein (1975, Nature 256:495-497), as well as 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) and the like are within the scope of the present
invention.
[0043] The monoclonal antibodies for diagnostic or therapeutic use
may be human monoclonal antibodies or chimeric human-mouse (or
other species) monoclonal antibodies. Human monoclonal antibodies
may be made by any of numerous techniques known in the art (e.g.,
Teng, et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:7308-7312;
Kozbor, et al., 1983, Immunology Today 4:72-79; Olsson, et al.,
1982, Meth. Enzymol. 92:3-16). Chimeric antibody molecules may be
prepared containing a mouse antigen-binding domain with human
constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci.
U.S.A. 81:6851, Takeda et al., 1985, Nature 314:452).
[0044] Various procedures known in the art may be used for the
production of polyclonal antibodies to epitopes of the Efl-6
described herein. For the production of antibody, various host
animals can be immunized by injection with the Efl-6, or a fragment
or derivative thereof, including but not limited to rabbits, mice,
rats, etc. Various adjuvants may be used to increase the
immunological response, depending on the host species, and
including but not limited to Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille
Calmette-Guerin) and Corynebacterium parvum.
[0045] A molecular clone of an antibody to a selected Efl-6 epitope
can be prepared by known techniques. Recombinant DNA methodology
(see e.g., Maniatis, et al., 1982, Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)
may be used to construct nucleic acid sequences which encode a
monoclonal antibody molecule, or antigen binding region
thereof.
[0046] Antibody molecules may be purified by known techniques,
e.g., immunoabsorption or immunoaffinity chromatography,
chromatographic methods such as HPLC (high performance liquid
chromatography), or a combination thereof, etc.
[0047] The present invention provides for antibody molecules as
well as fragments of such antibody molecules. Antibody fragments
which contain the idiotype of the molecule can be generated by
known techniques. For example, such fragments include but are not
limited to: the F(ab').sub.2 fragment which can be produced by
pepsin digestion of the antibody molecule; the Fab' fragments which
can be generated by reducing the disulfide bridges of the
F(ab').sub.2 fragment, and the Fab fragments which can be generated
by treating the antibody molecule with papain and a reducing
agent.
[0048] The present invention also provides for methods of treating
a patient suffering from a neurological disorder comprising
treating the patient with an effective amount of Efl-6, peptide
fragments thereof, or derivatives thereof capable of binding to Elk
receptor.
[0049] The Elk receptor is also expressed primarily in brain.
Accordingly, it is believed that the Elk binding ligand described
herein will support the induction of a differential function
and/or. influence the phenotype, such as growth and/or survival of
neural cells, expressing this receptor. As described in Gale, et
al., 1996, Oncogene 13:1343-1352, Elk-6 (described as Elk ligand 3
in the reference) is notable for its remarkable restricted and
prominent expression in the floor plate and roof plate of the
developing neural tube and its rhombomere-specific expression in
the developing hindbrain. This distribution suggests a role of
Efl-6 and its reciprocal receptor, in neuronal guidance and
boundary formation, critical features in the organization of the
developing vertebrate central nervous system.
[0050] The present invention also provides for pharmaceutical
compositions comprising the Efl-6 described herein, peptide
fragments thereof, or derivatives in a suitable pharmacologic
carrier.
[0051] The Efl-6 proteins, peptide fragments, or derivatives may be
administered systemically or locally. Any appropriate mode of
administration known in the art may be used, including, but not
limited to, intravenous, intrathecal, intraarterial, intranasal,
oral, subcutaneous, intraperitoneal, or by local injection or
surgical implant. Sustained release formulations are also provided
for.
[0052] As our understanding of neurodegenerative
disease/neurotrauma becomes clearer, it may become apparent that it
would be beneficial to decrease the effect of endogenous Efl-6.
Therefore, in areas of nervous system trauma, it may be desirable
to provide Efl-6 antagonists, including, but not limited to,
soluble forms of Efl-6 which may compete with cell-bound ligand for
interaction with Elk receptor. Alternatively, soluble forms of the
Elk receptors (e.g. expressed as "receptorbodies" produced as
described herein) may act as antagonists by binding, and thereby
inactivating the ligand. It may be desirable to provide such
antagonists locally at the injury site rather than systemically.
Use of an Efl-6 antagonist providing implant may be desirable.
[0053] Alternatively, certain conditions may benefit from an
increase in Efl-6 responsiveness. It may therefore be beneficial to
increase the number or binding affinity of Efl-6 in patients
suffering from such conditions. This could be achieved through gene
therapy using either Efl-6, Efl-6 expressing cells, or Elk receptor
or receptor chimeras (cells expressing the extracellular domain of
the Elk receptor). Selective expression of such recombinant
proteins in appropriate cells could be achieved using their
encoding genes controlled by tissue specific or inducible promoters
or by producing localized infection with replication defective
viruses carrying the recombinant genes.
[0054] The Efl-6 encoding DNA as deposited with the ATCC and having
accession number 97319 was isolated from a Stratagene (La Jolla,
Calif.) human brain (frontal cortex) library (Catalogue No.
936212). The library is in the .lambda.ZAPII vector. The sequence
of the Efl-6 coding region of this vector is set forth in FIG.
1.
[0055] Assays or purification of the Efl-6 protein may be conducted
by use of an Elk receptorbody, which consists of the extracellular
domain of Elk fused to the IgG1 constant region. This receptorbody
is prepared as follows: The Fc portion of human IgG1, starting from
the hinge region and extending to the carboxy terminus of the
molecule, was cloned from placental cDNA using PCR with
oligonucleotides corresponding to the published sequence of human
IgG1. Convenient restriction sites were also incorporated into the
oligonucleotides so as to allow cloning of the PCR fragment into an
expression vector. Expression vectors containing full length
receptors were modified either by restriction enzyme digests or by
PCR strategies so as to replace the transmembrane and intracellular
domains with restriction sites that allow cloning the human IgG1
fragment into these sites; this was done in such a way as to
generate a fusion protein with the receptor ectodomain as its
amino-terminus and the Fc portion of human IgG1 as its
carboxy-terminus. An alternative method of preparing receptorbodies
is described in Goodwin, et. al. 1993, Cell 73:447-456.
[0056] Deposit of Microorganisms
[0057] The following vector been deposited with the American Type
Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 in
accordance with the Budapest Treaty.
1 DEPOSIT ACCESSION NUMBER pBluescript SK-Efl-6 97319
[0058] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications
are intended to fall within the scope of the appended claims.
[0059] Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties.
Sequence CWU 1
1
3 1 1860 DNA Homo sapiens CDS (202)..(1224) Misc. feature
(166)...(166) Xaa is any amino acid 1 gaattcccac cccgggatct
gtgagactga gcgctctgcc gcgggggcgc gggcacagca 60 ggaarcaggt
ccgcgtgggc gctgggggca tcagctaccg gggtggtccg ggctgaagag 120
ccaggcagcc aaggcagcca ccccgggggg tgggcgactt tgggggagtt ggtgccccgc
180 cccccaggcc ttggcggggt c atg ggg ccc ccc cat tct ggg ccg ggg ggc
231 Met Gly Pro Pro His Ser Gly Pro Gly Gly 1 5 10 gtg cga gtc ggg
gcc ctg ctg ctg ctg ggg gtt ttg ggg ctg gtg tct 279 Val Arg Val Gly
Ala Leu Leu Leu Leu Gly Val Leu Gly Leu Val Ser 15 20 25 ggg ctc
agc ctg gag cct gtc tac tgg aac tcg gcg aat aag agg ttc 327 Gly Leu
Ser Leu Glu Pro Val Tyr Trp Asn Ser Ala Asn Lys Arg Phe 30 35 40
cag gca gag ggt ggt tat gtg ctg tac cct cag atc ggg gac cgg cta 375
Gln Ala Glu Gly Gly Tyr Val Leu Tyr Pro Gln Ile Gly Asp Arg Leu 45
50 55 gac ctg ctc tgc ccc cgg gcc cgg cct cct ggc cct cac tcc tct
cct 423 Asp Leu Leu Cys Pro Arg Ala Arg Pro Pro Gly Pro His Ser Ser
Pro 60 65 70 aat tat gag ttc tac aag ctg tac ctg gta ggg ggt gct
cag ggc cgg 471 Asn Tyr Glu Phe Tyr Lys Leu Tyr Leu Val Gly Gly Ala
Gln Gly Arg 75 80 85 90 cgc tgt gag gca ccc cct gcc cca aac ctc ctt
ctc act tgt gat cgc 519 Arg Cys Glu Ala Pro Pro Ala Pro Asn Leu Leu
Leu Thr Cys Asp Arg 95 100 105 cca gac ctg gat ctc cgc ttc acc atc
aag ttc cag gag tat agc cct 567 Pro Asp Leu Asp Leu Arg Phe Thr Ile
Lys Phe Gln Glu Tyr Ser Pro 110 115 120 aat ctc tgg ggc cac gag ttc
cgc tcg cac cac gat tac tac atc att 615 Asn Leu Trp Gly His Glu Phe
Arg Ser His His Asp Tyr Tyr Ile Ile 125 130 135 gcc aca tcg gat ggg
acc cgg gag ggc ctg gag agc ctg cag gga ggt 663 Ala Thr Ser Asp Gly
Thr Arg Glu Gly Leu Glu Ser Leu Gln Gly Gly 140 145 150 gtg tgc cta
acc aga ggc atg aag gtg ctt ctc cra gtg gga caa agt 711 Val Cys Leu
Thr Arg Gly Met Lys Val Leu Leu Xaa Val Gly Gln Ser 155 160 165 170
ccc cga gga ggg gct gtc ccc cga aaa cct gtg tct gaa atg ccc atg 759
Pro Arg Gly Gly Ala Val Pro Arg Lys Pro Val Ser Glu Met Pro Met 175
180 185 gaa aga gac cga ggg gca gcc cac agc ctg gag cct ggg aag gag
aac 807 Glu Arg Asp Arg Gly Ala Ala His Ser Leu Glu Pro Gly Lys Glu
Asn 190 195 200 ctg cca ggt gac ccc acc agc aat gca acc tcc cgg ggt
gct gaa ggc 855 Leu Pro Gly Asp Pro Thr Ser Asn Ala Thr Ser Arg Gly
Ala Glu Gly 205 210 215 ccc ctg ccc cct ccc agc atg cct gca gtg gct
ggg gca gca ggg ggg 903 Pro Leu Pro Pro Pro Ser Met Pro Ala Val Ala
Gly Ala Ala Gly Gly 220 225 230 ctg gcg ctg ctc ttg ctg ggc gtg gca
ggg gct ggg ggt gcc atg tgt 951 Leu Ala Leu Leu Leu Leu Gly Val Ala
Gly Ala Gly Gly Ala Met Cys 235 240 245 250 tgg cgg aga cgg cgg gcc
aag cct tcg gag agt cgc cac cct ggt cct 999 Trp Arg Arg Arg Arg Ala
Lys Pro Ser Glu Ser Arg His Pro Gly Pro 255 260 265 ggc tcc ttc ggg
agg gga ggg tct ctg ggc ctg ggg ggt gga ggt ggg 1047 Gly Ser Phe
Gly Arg Gly Gly Ser Leu Gly Leu Gly Gly Gly Gly Gly 270 275 280 atg
gga cct cgg gag gct gag cct ggg gag cta ggg ata gct ctg cgg 1095
Met Gly Pro Arg Glu Ala Glu Pro Gly Glu Leu Gly Ile Ala Leu Arg 285
290 295 ggt ggc ggg gct gca gat ccc ccc ttc tgc ccc cac tat gag aag
gtg 1143 Gly Gly Gly Ala Ala Asp Pro Pro Phe Cys Pro His Tyr Glu
Lys Val 300 305 310 agt ggt gac tat ggg cat cct gtg tat atc gtg cag
gat ggg ccc ccc 1191 Ser Gly Asp Tyr Gly His Pro Val Tyr Ile Val
Gln Asp Gly Pro Pro 315 320 325 330 cag agc cct cca aac atc tac tac
aag gta tga gggctcctct cacgtggcta 1244 Gln Ser Pro Pro Asn Ile Tyr
Tyr Lys Val 335 340 tcctgaatcc agcccttctt ggggtgctcc tccagtttaa
ttcctggttt gagggacacc 1304 tctaacatct cggccccctg tgccccccca
gccccttcac tcctcccggc tgctgtcctc 1364 gtctccactt ttaggattcc
ttaggattcc cactgcccca cttcctgccc tcccgtttgg 1424 ccatgggtgc
ccccctctgt ctcagtgtcc ctggatcctt tttccttggg gaggggcaca 1484
ggctcagcct cctctctgac catgacccag gcatccttgt ccccctcacc cacccagagc
1544 taggggcggg aacagcccac cttttggttg gcaccgcctt ctttctgcct
ctcactggtt 1604 ttctcttctc tatctcttat tctttccctc tcttccgtct
ctaggtctgt tcttcttccc 1664 tagcatcctc ctccccacat ctcctttcac
cctcttggct tcttatcctg tgnctctccc 1724 atctcctggg tgggggnatc
aaagcatttc tccccttagc tttcagcccc cttctganct 1784 ctcataccaa
ncactcccct cagtctgtca aaaatggggg gcttatgggg aagggtctga 1844
caatccaccc caggtc 1860 2 340 PRT Homo sapiens Misc. feature (166)
Xaa = unknown or other 2 Met Gly Pro Pro His Ser Gly Pro Gly Gly
Val Arg Val Gly Ala Leu 1 5 10 15 Leu Leu Leu Gly Val Leu Gly Leu
Val Ser Gly Leu Ser Leu Glu Pro 20 25 30 Val Tyr Trp Asn Ser Ala
Asn Lys Arg Phe Gln Ala Glu Gly Gly Tyr 35 40 45 Val Leu Tyr Pro
Gln Ile Gly Asp Arg Leu Asp Leu Leu Cys Pro Arg 50 55 60 Ala Arg
Pro Pro Gly Pro His Ser Ser Pro Asn Tyr Glu Phe Tyr Lys 65 70 75 80
Leu Tyr Leu Val Gly Gly Ala Gln Gly Arg Arg Cys Glu Ala Pro Pro 85
90 95 Ala Pro Asn Leu Leu Leu Thr Cys Asp Arg Pro Asp Leu Asp Leu
Arg 100 105 110 Phe Thr Ile Lys Phe Gln Glu Tyr Ser Pro Asn Leu Trp
Gly His Glu 115 120 125 Phe Arg Ser His His Asp Tyr Tyr Ile Ile Ala
Thr Ser Asp Gly Thr 130 135 140 Arg Glu Gly Leu Glu Ser Leu Gln Gly
Gly Val Cys Leu Thr Arg Gly 145 150 155 160 Met Lys Val Leu Leu Xaa
Val Gly Gln Ser Pro Arg Gly Gly Ala Val 165 170 175 Pro Arg Lys Pro
Val Ser Glu Met Pro Met Glu Arg Asp Arg Gly Ala 180 185 190 Ala His
Ser Leu Glu Pro Gly Lys Glu Asn Leu Pro Gly Asp Pro Thr 195 200 205
Ser Asn Ala Thr Ser Arg Gly Ala Glu Gly Pro Leu Pro Pro Pro Ser 210
215 220 Met Pro Ala Val Ala Gly Ala Ala Gly Gly Leu Ala Leu Leu Leu
Leu 225 230 235 240 Gly Val Ala Gly Ala Gly Gly Ala Met Cys Trp Arg
Arg Arg Arg Ala 245 250 255 Lys Pro Ser Glu Ser Arg His Pro Gly Pro
Gly Ser Phe Gly Arg Gly 260 265 270 Gly Ser Leu Gly Leu Gly Gly Gly
Gly Gly Met Gly Pro Arg Glu Ala 275 280 285 Glu Pro Gly Glu Leu Gly
Ile Ala Leu Arg Gly Gly Gly Ala Ala Asp 290 295 300 Pro Pro Phe Cys
Pro His Tyr Glu Lys Val Ser Gly Asp Tyr Gly His 305 310 315 320 Pro
Val Tyr Ile Val Gln Asp Gly Pro Pro Gln Ser Pro Pro Asn Ile 325 330
335 Tyr Tyr Lys Val 340 3 6 PRT Artificial Sequence Description of
Artificial Sequence peptide tag 3 His His His His His His 1 5
* * * * *