Note: Descriptions are shown in the official language in which they were submitted.
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BIOLOGICALLY ACTIVE EPH FAMILY LIGANDS
INTRODUCTION
' S
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
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
1 5 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 a1.,1990, Mol. Cell. Biol., 11: 5068-5078;
Fantl, et al., 1992, Cell, 69:413-413).
2 5 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,
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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).
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
1 5 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
2 0 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, CA: 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
2 5 bind this receptor (referred to herein as cognates). Thus, for
example, in adult mice, trkB was found to be preferentially
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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
1 0 development as well as play a role in the adult nervous system.
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,
2 0 and once signal transduction is initiated the cellular environment
will determine the phenotypic outcome of that signal transduction.
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
2 5 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.
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$: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-
974).
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,
2 0 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
2 5 member of this group of related receptors, Ehk-3 has been cloned
(Valenzuela, et al. 1995, Oncogene 10:1573-1580).
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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/CekS, Hek2/Sek4 and Htk receptors (Brambilla and Klein,
1995, Mol. Cell. Neurosci., 6:487-495, Gale, et al., 1996, Neuron
17:9-19).
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).
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
2 5 in development.
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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 W094/11384 (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
The present invention provides for a novel polypeptide
ligand, designated as Efl-6, that binds to the Elk, Nuk/CekS,
Hek2/Sek4, Htk, and Sek1 receptors on cells. More importantly, the
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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.
According to the invention, soluble forms .of the ligands
described herein may be used to promote biological responses in Elk,
Nuk/CekS, 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.
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.
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.
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BRIEF DESCRIPTION OF THE FIGURES
Figure 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
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/CekS, Hek2/Sek4 and Htk,
2 0 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.
The invention further provides a means of making
biologically active, soluble forms of the Efl-6 ligand, which are
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CA 02236001 2004-03-25
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.
The novel ligand described herein is designated as Efl (F.ph
transmembrane tyrosine kinase family ligands}-6. A deposit
designated as pbluescriptT"" SK-encoding Efl-6 was made with the
American Type Culture Collection on October 19, 1995 and has
received accession number 97319.
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.
2 0 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
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its cognate receptor may be used in the treatment of neurological or
other disorders.
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
1 0 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, eTa., by glycosylation, proteolytic
cleavage, linkage to an antibody molecule or other cellular ligand,
etc.
Cells that express Efl-6 may do so naturally or may be
genetically engineered to produce this ligand, as described su ra, by
transfection, transduction, electroporation, microinjection, via a
transgenic animal, etc. of nucleic acid encoding Efl-6 described
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herein in a suitable expression vector. A vector containing the cDNA
encoding for EFI-6 deposited with the American Type Culture
Collection under the terms of the Budapest Treaty on October 19,
1995 as pBluescriptSK'Efl-6 has been given the ATCC designation
97319.
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
1 0 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 Figure 1,
sequences of nucleic acids that hybridize to such sequence and which
1 5 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.
In addition, the present invention contemplates use of the
ligands described herein in soluble forms, truncated forms, and
20 tagged forms. This includes monomeric forms of the ligand which
may bind to the receptor and function as an antagonist.
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
2 5 transcriptional/translational control signals and the protein coding
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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-
1 5 797), the herpes thymidine kinase promoter (Wagner et al., 1981,
Proc. Natl. Acad. Sci. U.S.A. 78:144-1445), the regulatorysequences
of the metallothioein gene (Brinster al.,1982, Nature296:39-42);
et
prokaryotic expression vectors such the (i-lactamasepromoter
as
(Villa-Kamaroff, et al., 1978, Proc. Acad. 75:3727-
Natl. Sci.
U.S.A.
2 0 3731 ), or the tac promoter (DeBoer,al.,1983, Proc.
et 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)
25 promoter, alkaline phosphatase promoter, and the following animal
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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;
1 0 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
2 5 hormone gene control region which is active in the hypothalamus
(Mason et al., 1986, Science 234:1372-1378).
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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.
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
2 0 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 (eTa., thymidine kinase activity,
resistance to antibiotics, transformation phenotype, occlusion body
formation in baculovirus, etc.) caused by the insertion of foreign
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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.
Efl-6 appears to be a conventional transmembrane protein
with a cytoplasmic domain. The transmembrane domain is shown
underlined in Figure 1. Accordingly, the soluble or extracellular
1 5 domain of the ligand (sEfl-6) is encoded by the nucleotide sequence
from about nucleotide 274 to about nucleotide 873.
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
2 0 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
2 5 domain of the protein. The oligonucleotide spanning the C-terminus
region is modified so as to contain a stop codon prior to the
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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.
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.
In one embodiment, a "clustered" efl-6 is a dimer, made for
2 0 example, according to the present invention utilizing the Fc domain
of LgG (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
2 5 used to aggregate the ligands.
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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
1 0 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,
1 5 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
2 0 by epitopes to which antibodies are available, to allow for detection
on western blots, immunoprecipitation, or activity
depletion/blocking in bioassays.
Alternatively, multimers may be made by genetically
engineering and expressing molecules that consist of the soluble or
2 5 extracellular portion of the ligand followed by the Fc-domain of
hlgG, followed by either the c-jun or the c-fos leucine zippers
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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.
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
hlgG). Molecules may be engineered in which the length and
composition of the loop is varied, to allow for selection of
2 0 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
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comprises (soluble Efl)~, 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.
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
1 0 ligands, soluble forms of the ligands and clustered ligands may be
compared.
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.
2 0 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.
~ The recombinant factor may be purified by any technique
which allows for the subsequent formation of a stable, biologically
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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.
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
1 5 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 6418 resistance. Cells which lack an intact efl -6
may then be identified, e.g. by Southern blotting or Northern blotting
2 0 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
2 5 be used to define specific neuronal populations, or any other in vivo
processes, normally dependent upon the ligand.
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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.
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
1 5 "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc. pp. 77-
96) and the like are within the scope of the present invention.
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
2 0 may be made by any of numerous techniques known in the art (e.~c .,
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
2 5 regions (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851,
Takeda et al., 1985, Nature 314:452).
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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.
A molecular clone of an antibody to a selected Efl-6 epitope
1 5 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, New York)
may be used to construct nucleic acid sequences which encode a
monoclonal antibody molecule, or antigen binding region thereof.
2 0 Antibody molecules may be purified by known techniques,
immunoabsorption or immunoaffinity chromatography,
chromatographic methods such as HPLC (high performance liquid
chromatography), or a combination thereof, etc.
The present invention provides for antibody molecules as
2 5 well as fragments of such antibody molecules. Antibody fragments
which contain the idiotype of the molecule can be generated by
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known techniques. For example, such fragments include but are not
limited to: the F(ab')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')2 fragment,
and the Fab fragments which can be generated by treating the
antibody molecule with papain and a reducing agent.
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.
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
1 5 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
2 0 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.
The present invention also provides for pharmaceutical
2 5 compositions comprising the Efl-6 described herein, peptide
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WO 97/15667 PCT/US96/17201
fragments thereof, or derivatives in a suitable pharmacologic
carrier.
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.
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.
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
2 5 using either Efl-6, Efl-6 expressing cells, or Elk receptor or
receptor chimeras (cells expressing the extracellular domain of the
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WO 97/15667 PCT/US96/17201
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.
The Efl-6 encoding DNA as deposited with the ATCC and
having accession number 97319 was isolated from a Stratagene (La
Jolla, California) human brain (frontal cortex) library (Catalogue No.
936212). The library is in the ~,ZAPII vector. The sequence of the
Efl-6 coding region of this vector is set forth in Figure 1.
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 IgGI,
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
IgGI. 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
2 5 generate a fusion protein with the receptor ectodomain as its
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CA 02236001 2004-03-25
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.
p~p~(~' OF MtCROORt3AANISMS
The following vector been deposited with the American Type
Culture Collection, 12301 Parklewn Drive, Rockville, Maryland
20852 in accordance with the Budapest Treaty.
pBluescript SK-Efl-6 973 i 9
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
wilt 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.
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CA 02236001 1998-10-22
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: Regeneron Pharmaceuticals, Inc.
(ii) TITLE OF THE INVENTION: Biologically Active EPH
Family Ligands
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Osler, Hoskin & Harcourt
(B) STREET: 50 0'Connor Street, Suite 1500
(C) CITY: Ottawa
(D) STATE: ON
(E) COUNTRY: Canada
(F) ZIP: K1P 6L2
(v) COMPUTER READABLE FORM:
(A) COMPUTER: IBM Compatible
(B) OPERATING SYSTEM: DOS
(V) SOFTWARE: FastSEQ Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,236,001
(B) FILING DATE: 25-OCT-1996
(C) CLASSIFICATION: C12N-15/12
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: USSN 60/007,015
(B) FILING DATE: 25-OCT-1995
(viii) PATENT AGENT INFORMATION:
(A) NAME: David W. Aitken
(B) REFERENCE NUMBER: 12979
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1860 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: Efl-6
(B) LOCATION: 1...1860
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 202...1224
(D) OTHER INFORMATION: Coding region of putative signal
sequence from 202 to 273; coding region of mature
protein from 274 to 1224; coding region for
putative transmembrane from 874 to 948.
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CA 02236001 1998-10-22
(xi) SEQUENCE DESCRIPTION: N0:1:
SEQ
ID
GAATTCCCAC CCCGGGATCT GCGGGGGCGC GGGCACAGCA
60
GTGAGACTGA
GCGCTCTGCC
GGAARCAGGT CCGCGTGGGC GGGTGGTCCG GGCTGAAGAG
120
GCTGGGGGCA
TCAGCTACCG
CCAGGCAGCC AAGGCAGCCA TGGGGGAGTT GGTGCCCCGC
180
CCCCGGGGGG
TGGGCGACTT
CCCCCAGGCC TTGGCGGGGT ATGGGG CCCCCCCAT TCTGGGCCG GGGGGC 231
C
MetGly ProProHis SerGlyPro GlyGly
1 5 10
GTGCGAGTC GGGGCC CTGCTGCTG CTGGGGGTT TTGGGGCTG GTGTCT 279
ValArgVal GlyAla LeuLeuLeu LeuGlyVal LeuGlyLeu ValSer
15 20 25
GGGCTCAGC CTGGAG CCTGTCTAC TGGAACTCG GCGAATAAG AGGTTC 327
GlyLeuSer LeuGlu ProValTyr TrpAsnSer AlaAsnLys ArgPhe
30 35 40
CAGGCAGAG GGTGGT TATGTGCTG TACCCTCAG ATCGGGGAC CGGCTA 375
GlnAlaGlu GlyGly TyrValLeu TyrProGln IleGlyAsp ArgLeu
45 50 55
GACCTGCTC TGCCCC CGGGCCCGG CCTCCTGGC CCTCACTCC TCTCCT 423
AspLeuLeu CysPro ArgAlaArg ProProGly ProHisSer SerPro
60 65 70
AATTATGAG TTCTAC AAGCTGTAC CTGGTAGGG GGTGCTCAG GGCCGG 471
AsnTyrGlu PheTyr LysLeuTyr LeuValGly GlyAlaGln GlyArg
75 80 85 90
CGCTGTGAG GCACCC CCTGCCCCA AACCTCCTT CTCACTTGT GATCGC 519
ArgCysGlu AlaPro ProAlaPro AsnLeuLeu LeuThrCys AspArg
95 100 105
CCAGACCTG GATCTC CGCTTCACC ATCAAGTTC CAGGAGTAT AGCCCT 567
ProAspLeu AspLeu ArgPheThr IleLysPhe GlnGluTyr SerPro
110 115 120
AATCTCTGG GGCCAC GAGTTCCGC TCGCACCAC GATTACTAC ATCATT 615
AsnLeuTrp GlyHis GluPheArg SerHisHis AspTyrTyr IleIle
125 130 135
GCCACATCG GATGGG ACCCGGGAG GGCCTGGAG AGCCTGCAG GGAGGT 663
AlaThrSer AspGly ThrArgGlu GlyLeuGlu SerLeuGln GlyGly
140 145 150
GTGTGCCTA ACCAGA GGCATGAAG GTGCTTCTC CRAGTGGGA CAAAGT 711
ValCysLeu ThrArg GlyMetLys ValLeuLeu XaaValGly GlnSer
155 160 165 170
CCCCGAGGA GGGGCT GTCCCCCGA AAACCTGTG TCTGAAATG CCCATG 759
ProArgGly GlyAla ValProArg LysProVal SerGluMet ProMet
175 180 185
GAAAGAGAC CGAGGG GCAGCCCAC AGCCTGGAG CCTGGGAAG GAGAAC 807
GluArgAsp ArgGly AlaAlaHis SerLeuGlu ProGlyLys GluAsn
190 195 200
CTGCCAGGT GACCCC ACCAGCAAT GCAACCTCC CGGGGTGCT GAAGGC 855
LeuProGly AspPro ThrSerAsn AlaThrSer ArgGlyAla GluGly
205 210 215
CCCCTGCCC CCTCCC AGCATGCCT GCAGTGGCT GGGGCAGCA GGGGGG 903
ProLeuPro ProPro SerMetPro AlaValAla GlyAlaAla GlyGly
220 225 230
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CA 02236001 1998-10-22
CTG GCG CTG CTC TTG CTG GCA GGG GGG GGT GCC ATG 951
GGC GTG GCT TGT
Leu Ala Leu Leu Leu Leu Ala Gly Gly Gly Ala Met
Gly Val Ala Cys
235 240 245 250
TGG CGG AGA CGG CGG GCC TCG GAG CGC CAC CCT GGT 999
AAG CCT AGT CCT
Trp Arg Arg Arg Arg Ala Ser Glu Arg His Pro Gly
Lys Pro Ser Pro
255 260 265
GGC TCC TTC GGG AGG GGA CTG GGC GGG GGT GGA GGT 1047
GGG TCT CTG GGG
Gly Ser Phe Gly Arg Gly Leu Gly Gly Gly Gly Gly
Gly Ser Leu Gly
270 275 280
ATG GGA CCT CGG GAG GCT GGG GAG GGG ATA GCT CTG 1095
GAG CCT CTA CGG
Met Gly Pro Arg Glu Ala Gly Glu Gly Ile Ala Leu
Glu Pro Leu Arg
285 290 295
GGT GGC GGG GCT GCA GAT TTC TGC CAC TAT GAG AAG 1143
CCC CCC CCC GTG
Gly Gly Gly Ala Ala Asp Phe Cys His Tyr Glu Lys
Pro Pro Pro Val
300 305 310
AGT GGT GAC TAT GGG CAT TAT ATC CAG GAT GGG CCC 1191
CCT GTG GTG CCC
Ser Gly Asp Tyr Gly His Tyr Ile Gln Asp Gly Pro
Pro Val Val Pro
315 320 325 330
CAG AGC CCT CCA AAC ATC AAG GTA GGGCTC CTCTCACGTG 1244
TAC TAC TGA GCTA
Gln Ser Pro Pro Asn Ile Lys Val
Tyr Tyr
335 340
TCCTGAATCC AGCCCTTCTT GGGGTGCTCCTCCAGTTTAATTCCTGGTTT GAGGGACACC1304
TCTAACATCT CGGCCCCCTG TGCCCCCCCAGCCCCTTCACTCCTCCCGGC TGCTGTCCTC1364
GTCTCCACTT TTAGGATTCC TTAGGATTCCCACTGCCCCACTTCCTGCCC TCCCGTTTGG1424
CCATGGGTGC CCCCCTCTGT CTCAGTGTCCCTGGATCCTTTTTCCTTGGG GAGGGGCACA1484
GGCTCAGCCT CCTCTCTGAC CATGACCCAGGCATCCTTGTCCCCCTCACC CACCCAGAGC1544
TAGGGGCGGG AACAGCCCAC CTTTTGGTTGGCACCGCCTTCTTTCTGCCT CTCACTGGTT1604
TTCTCTTCTC TATCTCTTAT TCTTTCCCTCTCTTCCGTCTCTAGGTCTGT TCTTCTTCCC1664
TAGCATCCTC CTCCCCACAT CTCCTTTCACCCTCTTGGCTTCTTATCCTG TGNCTCTCCC1724
ATCTCCTGGG TGGGGGNATC AAAGCATTTCTCCCCTTAGCTTTCAGCCCC CTTCTGANCT1784
CTCATACCAA NCACTCCCCT CAGTCTGTCAAAAATGGGGGGCTTATGGGG AAGGGTCTGA1844
CAATCCACCC CAGGTC 1860
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 340 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID :
N0:2
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
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CA 02236001 1998-10-22
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
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: His6
(B) LOCATION: 1...6
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
His His His His His His
1 5
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