U.S. patent application number 09/897878 was filed with the patent office on 2002-11-07 for membrane associated protein zupar1.
Invention is credited to Bishop, Paul D., Gilbert, Teresa, Presnell, Scott R., Sheppard, Paul O..
Application Number | 20020164688 09/897878 |
Document ID | / |
Family ID | 22803010 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020164688 |
Kind Code |
A1 |
Sheppard, Paul O. ; et
al. |
November 7, 2002 |
Membrane associated protein zupar1
Abstract
Novel membrane associated polypeptides, polynucleotides encoding
those polypeptides, related compositions, antibodies and methods
are disclosed.
Inventors: |
Sheppard, Paul O.; (Granite
Falls, WA) ; Bishop, Paul D.; (Fall City, WA)
; Presnell, Scott R.; (Tacoma, WA) ; Gilbert,
Teresa; (Seattle, WA) |
Correspondence
Address: |
Phillip B.C. Jones, J.D., Ph.D.
ZymoGenetics, Inc
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Family ID: |
22803010 |
Appl. No.: |
09/897878 |
Filed: |
July 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60215446 |
Jun 30, 2000 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/183; 435/320.1; 435/325; 435/6.16; 530/350; 536/23.2 |
Current CPC
Class: |
C07K 14/52 20130101;
C07K 2319/00 20130101; C07K 14/47 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 435/183; 530/350; 536/23.2; 435/6 |
International
Class: |
C12P 021/02; C12N
005/06; C07H 021/04; C12N 009/00; C07K 014/435; C12Q 001/68 |
Claims
I claim:
1. An isolated polypeptide, comprising an amino acid sequence of at
least 15 contiguous amino acid residues of SEQ ID NO:2.
2. The isolated polypeptide of claim 1, wherein the isolated
polypeptide comprises at least 20 contiguous amino acid residues of
SEQ ID NO:2.
3. The isolated polypeptide of claim 1, wherein the isolated
polypeptide comprises at least 30 contiguous amino acid residues of
SEQ ID NO:2.
4. The isolated polypeptide of claim 1, wherein the isolated
polypeptide is selected from the group consisting of: a) the
polypeptide of SEQ ID NO:6; b) the polypeptide of SEQ ID NO:7; or
c) the polypetide of SEQ ID NO:8.
5. The isolated polypeptide of claim 1, wherein the isolated
polypeptide is selected from the group consisting of: a) the
polypeptide of SEQ ID NO:9; b) the polypeptide of SEQ ID NO:10; or
c) the polypetide of SEQ ID NO:11.
6. The isolated polypeptide of claim 1, comprising the amino acid
sequence of SEQ ID NO:2.
7. An isolated nucleic acid molecule, wherein the nucleic acid
molecule is selected from the group consisting of (a) a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:3,
(b) a nucleic acid molecule encoding the amino acid sequence of SEQ
ID NO:2, and (c) a nucleic acid molecule that remains hybridized
following stringent wash conditions to a nucleic acid molecule
consisting of a nucleotide sequence of SEQ ID NO:1, or the
complement of a nucleotide sequence of SEQ ID NO:1.
8. The isolated nucleic acid molecule of claim 7, wherein any
difference between the amino acid sequence encoded by the nucleic
acid molecule and the corresponding amino acid sequence of SEQ ID
NO:2 is due to a conservative amino acid substitution.
9. The isolated nucleic acid molecule of claim 7, comprising the
nucleotide sequence of SEQ ID NO:1.
10. A vector, comprising the isolated nucleic acid molecule of
claim 9.
11. An expression vector, comprising the isolated nucleic acid
molecule of claim 9, a transcription promoter, and a transcription
terminator, wherein the promoter is operably linked with the
nucleic acid molecule, and wherein the nucleic acid molecule is
operably linked with the transcription terminator.
12. A recombinant host cell comprising the expression vector of
claim 11, wherein the host cell is selected from the group
consisting of bacterium, yeast cell, fungal cell, insect cell,
avian cell, mammalian cell, and plant cell.
13. A method of using the expression vector of claim 11 to produce
zupar1 protein, comprising culturing recombinant host cells that
comprise the expression vector and that produce the zupar1
protein.
14. The method of claim 13, further comprising isolating the zupar1
protein from the cultured recombinant host cells.
15. An antibody or antibody fragment that specifically binds with
the polypeptide of claim 6.
16. The antibody of claim 15, wherein the antibody is selected from
the group consisting of: (a) polyclonal antibody, (b) murine
monoclonal antibody, (c) humanized antibody derived from (b), and
(d) human monoclonal antibody.
17. An anti-idiotype antibody that specifically the binds the
antibody of claim 15.
18. A method of detecting the presence of zupar1 gene expression in
a biological sample, comprising: (a) contacting a zupar1 nucleic
acid probe under hybridizing conditions with either (i) test RNA
molecules isolated from the biological sample, or (ii) nucleic acid
molecules synthesized from the isolated RNA molecules, wherein the
probe consists of a nucleotide sequence comprising a portion of the
nucleotide sequence of the nucleic acid molecule of claim 8, or
complements thereof, and (b) detecting the formation of hybrids of
the nucleic acid probe and either the test RNA molecules or the
synthesized nucleic acid molecules, wherein the presence of the
hybrids indicates the presence of zupar1 RNA in the biological
sample, or, (a') contacting the biological sample with an antibody,
or an antibody fragment, which specifically binds with a
polypeptide consisting of the amino acid sequence of SEQ ID NO:2,
wherein the contacting is performed under conditions that allow the
binding of the antibody or antibody fragment to the biological
sample, and (b') detecting any of the bound antibody or bound
antibody fragment.
19. A composition, comprising a carrier and a polypeptide selected
from the group consisting of: a) the polypeptide of SEQ ID NO:6; b)
the polypeptide of SEQ ID NO:7; c) the polypetide of SEQ ID NO:8;
d) the polypeptide of SEQ ID NO:9; e) the polypeptide of SEQ ID
NO:10; or f) the polypetide of SEQ ID NO:11.
20. A fusion protein, comprising a polypeptide selected from the
group consisting of: a) the polypeptide of SEQ ID NO:6; b) the
polypeptide of SEQ ID NO:7; c) the polypeptide of SEQ ID NO:8; d)
the polypeptide of SEQ ID NO:9; e) the polypeptide of SEQ ID NO:10;
or f) the polypetide of SEQ ID NO:11.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to Provisional Application
60/215,446, filed on Jun. 30, 2000. Under 35 U.S.C.
.sctn.119(e)(1), this application claims benefit of said
Provisional Application.
TECHNICAL FIELD
[0002] The present invention relates generally to a gene that
encodes a new human protein. In particular, the present invention
relates to a novel protein, designated "zupar1," and to nucleic
acid molecules encoding zupar1.
BACKGROUND OF THE INVENTION
[0003] Membrane associated proteins are found bound to the membrane
face, anchored to membrane, or extending entirely through the
membrane one or more times, with domains exposed both extra- and
intracellularly. Membrane proteins act intracellularly,
extracellularly, or pass signals or molecules though the membrane.
Membrane associated proteins can also be disassociated from the
membrane by solublization or truncation, for example, the soluble
form used like a secreted protein. Ligands and receptors can be
found associated to the membrane surface. Membrane proteins act to
protect the cell from foreign substances, modulate proliferation
and differentiation, transmit signals, and transport ions. Membrane
proteins includes major histocompatibility complexes, plasminogen
activators, glycoproteins, GPI-anchored proteins, and the like.
[0004] Many of these membrane associated polypeptides are useful
therapeutic and diagnostic agents. A continuing need, therefore,
exists for the discovery and characterization of new membrane
associated proteins. The present invention addresses this need by
providing a novel human membrane associated proteins and related
compositions and methods.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention provides a novel human secreted
protein, designated "zupar1." The present invention also provides
zupar1 variant polypeptides and zupar1 fusion proteins, as well as
nucleic acid molecules encoding such polypeptides and proteins, and
methods for using these nucleic acid molecules and amino acid
sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0006] 1. Overview
[0007] The present invention provides nucleic acid molecules that
encode a new protein, designated as "zupar1." An illustrative
nucleotide sequence that encodes zupar1 is provided by SEQ ID NO:1.
The encoded polypeptide comprises the following amino acid
sequence: MVLCWLLLLV MALPPGTTGV KDCVFCELTD SMQCPGTYMH CGDDEDCFTG
HGVAPGTGPV INKGCLRATS CGLEEPVSYR GVTYSLTTNC CTGALCNRAP SSQTVGATTS
LALGLGMLLP PRLL (SEQ ID NO:2). Thus, the zupar1 nucleotide sequence
described herein encodes a polypeptide of 124 amino acids, as shown
in SEQ ID NO:2. Zupar1 gene is located at 19q.13.32.
[0008] As detailed below, the present invention includes
polypeptides, comprising an amino acid sequence of at least 15
contiguous amino acid residues of SEQ ID NO:2. The present
invention further includes polypeptides comprising an amino acid
sequence of at least 20, or 30, contiguous amino acid residues of
SEQ ID NO:2. Such polypeptides include those of SEQ ID
NOs:5-10.
[0009] The present invention further provides antibodies and
antibody fragments that specifically bind with such polypeptides.
Exemplary antibodies include polyclonal antibodies, murine
monoclonal antibodies, humanized antibodies derived from murine
monoclonal antibodies, and human monoclonal antibodies.
Illustrative antibody fragments include F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, scFv, and minimal recognition units. The present
invention also includes anti-idiotype antibodies that specifically
bind with such antibodies or antibody fragments. The present
invention further includes compositions comprising a carrier and a
peptide, polypeptide, antibody, or anti-idiotype antibody described
herein.
[0010] The present invention also provides isolated nucleic acid
molecules that encode a zupar1 polypeptide, wherein the nucleic
acid molecule is selected from the group consisting of (a) a
nucleic acid molecule comprising the nucleotide sequence of SEQ ID
NO:3, (b) a nucleic acid molecule encoding the amino acid sequence
of SEQ ID NO:2, and (c) a nucleic acid molecule that remains
hybridized following stringent wash conditions to a nucleic acid
molecule consisting of a nucleotide sequence, or the complement of
the nucleotide sequence, of SEQ ID NO:1.
[0011] Illustrative nucleic acid molecules include those in which
any difference between the amino acid sequence encoded by the
nucleic acid molecule and the corresponding amino acid sequence of
SEQ ID NO:2 is due to a conservative amino acid substitution. The
present invention further contemplates isolated nucleic acid
molecules that comprise the nucleotide sequence of SEQ ID NO:1.
[0012] The present invention also includes vectors and expression
vectors comprising such nucleic acid molecules. Such expression
vectors may comprise a transcription promoter, and a transcription
terminator, wherein the promoter is operably linked with the
nucleic acid molecule, and wherein the nucleic acid molecule is
operably linked with the transcription terminator. The present
invention further includes recombinant host cells comprising these
vectors and expression vectors. Illustrative host cells include
bacterial, yeast, fungal, insect, avian, mammalian, and plant
cells. Recombinant host cells comprising such expression vectors
can be used to produce zupar1 polypeptides by culturing such
recombinant host cells that comprise the expression vector and that
produce the zupar1 protein, and, optionally, isolating the zupar1
protein from the cultured recombinant host cells.
[0013] The present invention also contemplates methods for
detecting the presence of zupar1 RNA in a biological sample,
comprising the steps of (a) contacting a zupar1 nucleic acid probe
under hybridizing conditions with either (i) test RNA molecules
isolated from the biological sample, or (ii) nucleic acid molecules
synthesized from the isolated RNA molecules, wherein the probe has
a nucleotide sequence comprising a portion of the nucleotide
sequence of SEQ ID NO:1, or its complement, and (b) detecting the
formation of hybrids of the nucleic acid probe and either the test
RNA molecules or the synthesized nucleic acid molecules, wherein
the presence of the hybrids indicates the presence of zupar1 RNA in
the biological sample.
[0014] The present invention further provides methods for detecting
the presence of zupar1 polypeptide in a biological sample,
comprising the steps of: (a) contacting the biological sample with
an antibody or an antibody fragment that specifically binds with a
polypeptide having the amino acid sequence of SEQ ID NO:2, wherein
the contacting is performed under conditions that allow the binding
of the antibody or antibody fragment to the biological sample, and
(b) detecting any of the bound antibody or bound antibody fragment.
Such an antibody or antibody fragment may further comprise a
detectable label selected from the group consisting of
radioisotope, fluorescent label, chemiluminescent label, enzyme
label, bioluminescent label, and colloidal gold. An exemplary
biological sample is a human biological sample, such as a biopsy or
autopsy specimen.
[0015] The present invention also provides kits for performing
these detection methods. For example, a kit for detection of zupar1
gene expression may comprise a container that comprises a nucleic
acid molecule, wherein the nucleic acid molecule is selected from
the group consisting of (a) a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:1, (b) a nucleic acid molecule
comprising the complement of the nucleotide sequence of SEQ ID
NO:1, (c) a nucleic acid molecule that is a fragment of (a)
consisting of at least eight nucleotides, and (d) a nucleic acid
molecule that is a fragment of (b) consisting of at least eight
nucleotides. Such a kit may also comprise a second container that
comprises one or more reagents capable of indicating the presence
of the nucleic acid molecule. On the other hand, a kit for
detection of zupar1 protein may comprise a container that comprises
an antibody, or an antibody fragment, that specifically binds with
a polypeptide having the amino acid sequence of SEQ ID NO:2.
[0016] The present invention further provides fusion proteins
comprising a zupar1 polypeptide and an immunoglobulin moiety. In
such fusion proteins, the immunoglobulin moiety may be an
immunoglobulin heavy chain constant region, such as a human F.sub.C
fragment. The present invention further includes isolated nucleic
acid molecules that encode such fusion proteins.
[0017] The present invention also provides a carrier and a zupar1
polypeptide.
[0018] These and other aspects of the invention will become evident
upon reference to the following detailed description. In addition,
various references are identified below and are incorporated by
reference in their entirety.
[0019] 2. Definitions
[0020] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0021] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally-occurring nucleotides (such as DNA and RNA), or analogs
of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0022] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
[0023] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons as
compared to a reference nucleic acid molecule that encodes a
polypeptide. Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0024] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0025] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0026] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0027] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0028] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0029] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements (DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars
in Cancer Biol. 1:47 (1990)), glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye
et al., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)). If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0030] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0031] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
regulatory element may contain a nucleotide sequence that binds
with cellular factors enabling transcription exclusively or
preferentially in particular cells, tissues, or organelles. These
types of regulatory elements are normally associated with genes
that are expressed in a "cell-specific," "tissue-specific," or
"organelle-specific" manner.
[0032] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0033] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0034] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides."
[0035] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0036] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0037] An "integrated genetic element" is a segment of DNA that has
been incorporated into a chromosome of a host cell after that
element is introduced into the cell through human manipulation.
Within the present invention, integrated genetic elements are most
commonly derived from linearized plasmids that are introduced into
the cells by electroporation or other techniques. Integrated
genetic elements are passed from the original host cell to its
progeny.
[0038] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, that has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide tetracycline resistance
or ampicillin resistance.
[0039] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0040] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. In the present context, an example of a recombinant host is
a cell that produces zupar1 from an expression vector. In contrast,
zupar1 can be produced by a cell that is a "natural source" of
zupar1, and that lacks an expression vector.
[0041] "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0042] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two genes. For example, a fusion protein can comprise at least part
of a zupar1 polypeptide fused with a polypeptide that binds an
affinity matrix. Such a fusion protein provides a means to isolate
large quantities of zupar1 using affinity chromatography.
[0043] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule termed a "ligand." This interaction
mediates the effect of the ligand on the cell. Receptors can be
membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid
stimulating hormone receptor, beta-adrenergic receptor) or
multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3
receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor
and IL-6 receptor). Membrane-bound receptors are characterized by a
multi-domain structure comprising an extracellular ligand-binding
domain and an intracellular effector domain that is typically
involved in signal transduction. In certain membrane-bound
receptors, the extracellular ligand-binding domain and the
intracellular effector domain are located in separate polypeptides
that comprise the complete functional receptor.
[0044] In general, the binding of ligand to receptor results in a
conformational change in the receptor that causes an interaction
between the effector domain and other molecule(s) in the cell,
which in turn leads to an alteration in the metabolism of the cell.
Metabolic events that are often linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, increases in cyclic AMP production, mobilization
of cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids.
[0045] The term "secretory signal sequence" denotes a nucleotide
sequence that encodes a peptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0046] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, or greater than 99%
pure. One way to show that a particular protein preparation
contains an isolated polypeptide is by the appearance of a single
band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of the protein preparation and Coomassie Brilliant
Blue staining of the gel. However, the term "isolated" does not
exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0047] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0048] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0049] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a polypeptide encoded by a
splice variant of an mRNA transcribed from a gene.
[0050] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, co-stimulatory
molecules, hematopoietic factors, and synthetic analogs of these
molecules.
[0051] An "anti-idiotype antibody" is an antibody that binds with
the variable region domain of an immunoglobulin. In the present
context, an anti-idiotype antibody binds with the variable region
of an anti-zupar1 antibody, and thus, an anti-idiotype antibody
mimics an epitope of zupar1.
[0052] An "antibody fragment" is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-zupar1
monoclonal antibody fragment binds with an epitope of zupar1.
[0053] The term "antibody fragment" also includes a synthetic or a
genetically engineered polypeptide that binds to a specific
antigen, such as polypeptides consisting of the light chain
variable region, "Fv" fragments consisting of the variable regions
of the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker ("scFv proteins"), and minimal recognition
units consisting of the amino acid residues that mimic the
hypervariable region.
[0054] A "chimeric antibody" is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0055] "Humanized antibodies" are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain.
[0056] As used herein, a "therapeutic agent" is a molecule or atom
which is conjugated to an antibody moiety to produce a conjugate
which is useful for therapy. Examples of therapeutic agents include
drugs, toxins, immunomodulators, chelators, boron compounds,
photoactive agents or dyes, and radioisotopes.
[0057] A "detectable label" is a molecule or atom, which can be
conjugated to an antibody moiety to produce a molecule useful for
diagnosis. Examples of detectable labels include chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic
ions, or other marker moieties.
[0058] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075 (1985); Nilsson et al.,
Methods Enzymol. 198:3 (1991)), glutathione S transferase (Smith
and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer
et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)), substance P,
FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2:95 (1991). Nucleic acid molecules encoding affinity
tags are available from commercial suppliers (e.g., Pharmacia
Biotech, Piscataway, N.J.).
[0059] A "naked antibody" is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized antibodies.
[0060] As used herein, the term "antibody component" includes both
an entire antibody and an antibody fragment.
[0061] An "immunoconjugate" is a conjugate of an antibody component
with a therapeutic agent or a detectable label.
[0062] As used herein, the term "antibody fusion protein" refers to
a recombinant molecule that comprises an antibody component and a
therapeutic agent. Examples of therapeutic agents suitable for such
fusion proteins include immunomodulators ("antibody-immunomodulator
fusion protein") and toxins ("antibody-toxin fusion protein").
[0063] An "antigenic peptide" is a peptide, which will bind a major
histocompatibility complex molecule to form an MHC-peptide complex,
which is recognized by a T cell, thereby inducing a cytotoxic
lymphocyte response upon presentation to the T cell. Thus,
antigenic peptides are capable of binding to an appropriate major
histocompatibility complex molecule and inducing a cytotoxic T
cells response, such as cell lysis or specific cytokine release
against the target cell which binds or expresses the antigen. The
antigenic peptide can be bound in the context of a class I or class
II major histocompatibility complex molecule, on an antigen
presenting cell or on a target cell.
[0064] An "anti-sense RNA or anti-sense gene" In eukaryotes, RNA
polymerase II catalyzes the transcription of a structural gene to
produce mRNA. A nucleic acid molecule can be designed to contain an
RNA polymerase II template in which the RNA transcript has a
sequence that is complementary to that of a specific mRNA. The RNA
transcript is termed an "anti-sense RNA" and a nucleic acid
molecule that encodes the anti-sense RNA is termed an "anti-sense
gene." Anti-sense RNA molecules are capable of binding to mRNA
molecules, resulting in an inhibition of mRNA translation.
[0065] An "anti-sense oligonucleotide specific for zupar1" or an
"zupar1 anti-sense oligonucleotide" is an oligonucleotide having a
sequence (a) capable of forming a stable triplex with a portion of
the zupar1 gene, or (b) capable of forming a stable duplex with a
portion of an mRNA transcript of the zupar1 gene.
[0066] A "ribozyme" is a nucleic acid molecule that contains a
catalytic center. The term includes RNA enzymes, self-splicing
RNAs, self-cleaving RNAs, and nucleic acid molecules that perform
these catalytic functions. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene."
[0067] An "external guide sequence" is a nucleic acid molecule that
directs the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, resulting in the cleavage of the mRNA by
RNase P. A nucleic acid molecule that encodes an external guide
sequence is termed an "external guide sequence gene."
[0068] The term "variant zupar1 gene" refers to nucleic acid
molecules that encode a polypeptide having an amino acid sequence
that is a modification of SEQ ID NO:2. Such variants include
naturally-occurring polymorphisms of zupar1 genes, as well as
synthetic genes that contain conservative amino acid substitutions
of the amino acid sequence of SEQ ID NO:2. Additional variant forms
of zupar1 genes are nucleic acid molecules that contain insertions
or deletions of the nucleotide sequences described herein. A
variant zupar1 gene can be identified by determining whether the
gene hybridizes with a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:1, or its complement, under stringent
conditions.
[0069] Alternatively, variant zupar1 genes can be identified by
sequence comparison. Two amino acid sequences have "100% amino acid
sequence identity" if the amino acid residues of the two amino acid
sequences are the same when aligned for maximal correspondence.
Similarly, two nucleotide sequences have "100% nucleotide sequence
identity" if the nucleotide residues of the two nucleotide
sequences are the same when aligned for maximal correspondence.
Sequence comparisons can be performed using standard software
programs such as those included in the LASERGENE bioinformatics
computing suite, which is produced by DNASTAR (Madison, Wis.).
Other methods for comparing two nucleotide or amino acid sequences
by determining optimal alignment are well-known to those of skill
in the art (see, for example, Peruski and Peruski, The Internet and
the New Biology: Tools for Genomic and Molecular Research (ASM
Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and
Computer Databases of Nucleic Acids and Proteins," in Methods in
Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and
Bishop (ed.), Guide to Human Genome Computing, 2nd Edition
(Academic Press, Inc. 1998)). Particular methods for determining
sequence identity are described below.
[0070] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0071] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0072] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0073] The present invention includes functional fragments of
zupar1 genes. Within the context of this invention, a "functional
fragment" of a zupar1 gene refers to a nucleic acid molecule that
encodes a portion of a zupar1 polypeptide, which either
specifically binds with an anti-zupar1 antibody or displays
biological activity as discussed herein. For example, a functional
fragment of a zupar1 gene described herein comprises a portion of
the nucleotide sequence of SEQ ID NO:1, and encodes a polypeptide
that specifically binds with an anti- zupar1 antibody.
[0074] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are understood to be
approximate values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to .+-.10%.
[0075] 3. Production of a Nucleic Acid Molecule That Encodes
zupar1
[0076] The 375 bp zupar1 nucleic acid sequence (SEQ ID NO:1)
encodes a 124 amino acid residue polypeptide (SEQ ID NO:2) having a
signal sequence (amino acid residues 1-18 of SEQ ID NO:2, encoded
by nucleic acids 1-54 of SEQ ID NO:1, or amino acid residues 1-19
of SEQ ID NO:2, encoded by nucleic acids 1-57 of SEQ ID NO:1), an
extracellular domain (amino acid residues 19 or 20-110 of SEQ ID
NO:2, encoded by nucleic acids 1-54 or 57-330 of SEQ ID NO:1), and
a membrane associated domain (amino acid residues 111-124 of SEQ ID
NO:2, encoded by 331-372 of SEQ ID NO:1). zupar1 shares some
homology with the soluble and membrane-bound GPI-anchored proteins
CD59 (Davies et al., J. Exp. Med. 170:637-54, 1989; Okada et al.,
Biochem. Biophys. Res. Commun. 162:1553-59, 1989; Surita et al., J.
Biochem. 106:555-7, 1989; and Swada et al., Nucleic Acids Res.
17:6728, 1989), and Vasopressin-neurophysin 2-copeptin precursor
(Chauvet et al., Proc. Natl. Acad. Sci. USA 80:2839-43, 1983; and
Seidah et al., Biochem. Biophys. Res. Comm. 100:901-7, 1981),
Vasotocin-neurophysin VT precursor (Nojiri et al., Proc. Natl.
Acad. Sci. USA 84:3043-46, 1987), and Urokinase plasminogen
activator surface receptor precursor (Roldan et al., EMBO J.
9:467-74, 1990; and Behrendt et al., J. Biol. Chem. 265:6453-60,
1990).
[0077] Zupar1, as represented by SEQ ID NO:2, has five beta strands
(amino acid residues 22-24, 38-40, 46-52, and 60-66, 89-91, of SEQ
ID NO:2, encoded by nucleic acids 64-72, 112-120, 136-156, 178-198,
265-273, of SEQ ID NO:1), and a single alpha helix (amino acid
residues 76-80, of SEQ ID NO:2, encoded by nucleic acids 226-240 of
SEQ ID NO:1). This is similar to the secondary structure of CD59
(Fletcher et al., Structure 2:185, 1994). zupar1 also has 10
cysteine residues (23, 26, 34, 41, 47, 65, 71, 90, 91, and 95 of
SEQ ID NO:2) that form 5 disulfide bonds. The pairing of the
residues is: residues 23-47, residues 26-34, residues 41-65,
residues 71-90, and residues 91-95 of SEQ ID NO:2. The arginine
residue at position 80 of SEQ ID NO:2, is considered an active side
residue.
[0078] Another aspect of the present invention includes zupar1
polypeptide fragments. Preferred fragments include soluble forms of
the protein that do not contain the membrane associated segment
from amino acid residues 111-124 of SEQ ID NO:2, fragments
containing the active site residue, amino acid residue 80 of SEQ ID
NO:2, fragments containing some or all of the cysteine residues
found at amino acid residues 23, 26, 34, 41, 47, 65, 71, 90, 91,
and 95 of SEQ ID NO:2, fragments with and with out the signal
sequence, amino acid residues 1-19 or 20 of SEQ ID NO:2.
Polypeptide fragments also include those sequences found in SEQ ID
NOs:5-10. Such fragments may include +/-6 additional amino acid
residues at either or both the N and C-terminus for protein
engineering and stability.
[0079] These fragments are particularly useful in competing with
complement or other membrane targeted complexes in reducing
inflammation, coagulation, or tissue remodeling. Such fragments
would be useful in a variety of autoimmune diseases, circulatory
anomalies, and blocking tissue remodeling in aggressive cancers.
Polynucleotides encoding such fragments are also encompassed by the
present invention.
[0080] Electronic analysis of the tissue distribution of the mRNA
corresponding to this novel DNA showed that zupar1 is strongly
represented in testis and was also found in germ cell tumors. PCR
was carried out and detected zupar1 expression in kidney, liver,
lung, pancreas, placenta, colon, prostate, small intestine, testis,
and tonsil.
[0081] Nucleic acid molecules encoding a human zupar1 amino acid
sequence can be obtained by screening a human cDNA or genomic
library using polynucleotide probes based upon SEQ ID NO:1. These
techniques are standard and well-established.
[0082] As an illustration, a nucleic acid molecule that encodes a
human zupar1 gene can be isolated from a human cDNA library. In
this case, the first step would be to prepare the cDNA library by
isolating RNA from tissue using methods well-known to those of
skill in the art. In general, RNA isolation techniques must provide
a method for breaking cells, a means of inhibiting RNase-directed
degradation of RNA, and a method of separating RNA from DNA,
protein, and polysaccharide contaminants. For example, total RNA
can be isolated by freezing tissue in liquid nitrogen, grinding the
frozen tissue with a mortar and pestle to lyse the cells,
extracting the ground tissue with a solution of phenol/chloroform
to remove proteins, and separating RNA from the remaining
impurities by selective precipitation with lithium chloride (see,
for example, Ausubel et al. (eds.), Short Protocols in Molecular
Biology, 3.sup.rd Edition, pages 4-1 to 4-6 (John Wiley & Sons
1995) ["Ausubel (1995)"]; Wu et al., Methods in Gene Biotechnology,
pages 33-41 (CRC Press, Inc. 1997) ["Wu (1997)"]). Alternatively,
total RNA can be isolated from tissue by extracting ground tissue
with guanidinium isothiocyanate, extracting with organic solvents,
and separating RNA from contaminants using differential
centrifugation (see, for example, Chirgwin et al., Biochemistry
18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at
pages 33-41). In order to construct a cDNA library, poly(A).sup.+
RNA must be isolated from a total RNA preparation.
[0083] Poly(A).sup.+ RNA can be isolated from total RNA using the
standard technique of oligo(dT)-cellulose chromatography (see, for
example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972);
Ausubel (1995) at pages 4-11 to 4-12).
[0084] Double-stranded cDNA molecules are synthesized from
poly(A).sup.+ RNA using techniques well-known to those in the art.
(see, for example, Wu (1997) at pages 41-46). Moreover,
commercially available kits can be used to synthesize
double-stranded cDNA molecules. For example, such kits are
available from Life Technologies, Inc. (Gaithersburg, Md.),
CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Promega
Corporation (Madison, Wis.) and STRATAGENE (La Jolla, Calif.).
[0085] Various cloning vectors are appropriate for the construction
of a cDNA library. For example, a cDNA library can be prepared in a
vector derived from bacteriophage, such as a .lambda.gt10 vector.
See, for example, Huynh et al., "Constructing and Screening cDNA
Libraries in .lambda.gt10 and .lambda.gt11," in DNA Cloning: A
Practical Approach Vol. I, Glover (ed.), page 49 (IRL Press, 1985);
Wu (1997) at pages 47-52.
[0086] Alternatively, double-stranded cDNA molecules can be
inserted into a plasmid vector, such as a PBLUESCRIPT vector
(STRATAGENE; La Jolla, Calif.), a LAMDAGEM-4 (Promega Corp.) or
other commercially available vectors. Suitable cloning vectors also
can be obtained from the American Type Culture Collection
(Manassas, Va.).
[0087] To amplify the cloned cDNA molecules, the cDNA library is
inserted into a prokaryotic host, using standard techniques. For
example, a cDNA library can be introduced into competent E. coli
DH5 cells, which can be obtained, for example, from Life
Technologies, Inc. (Gaithersburg, Md.).
[0088] A human genomic library can be prepared by means well-known
in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6;
Wu (1997) at pages 307-327). Genomic DNA can be isolated by lysing
tissue with the detergent Sarkosyl, digesting the lysate with
proteinase K, clearing insoluble debris from the lysate by
centrifugation, precipitating nucleic acid from the lysate using
isopropanol, and purifying resuspended DNA on a cesium chloride
density gradient.
[0089] DNA fragments that are suitable for the production of a
genomic library can be obtained by the random shearing of genomic
DNA or by the partial digestion of genomic DNA with restriction
endonucleases. Genomic DNA fragments can be inserted into a vector,
such as a bacteriophage or cosmid vector, in accordance with
conventional techniques, such as the use of restriction enzyme
digestion to provide appropriate termini, the use of alkaline
phosphatase treatment to avoid undesirable joining of DNA
molecules, and ligation with appropriate ligases. Techniques for
such manipulation are well-known in the art (see, for example,
Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages
307-327).
[0090] Nucleic acid molecules that encode a human zupar1 gene can
also be obtained using the polymerase chain reaction (PCR) with
oligonucleotide primers having nucleotide sequences that are based
upon the nucleotide sequences of the human zupar1 gene, as
described herein. General methods for screening libraries with PCR
are provided by, for example, Yu et al., "Use of the Polymerase
Chain Reaction to Screen Phage Libraries," in Methods in Molecular
Biology, Vol. 15: PCR Protocols: Current Methods and Applications,
White (ed.), pages 211-215 (Humana Press, Inc. 1993). Moreover,
techniques for using PCR to isolate related genes are described by,
for example, Preston, "Use of Degenerate Oligonucleotide Primers
and the Polymerase Chain Reaction to Clone Gene Family Members," in
Methods in Molecular Biology, Vol. 15: PCR Protocols: Current
Methods and Applications, White (ed.), pages 317-337 (Humana Press,
Inc. 1993).
[0091] Alternatively, human genomic libraries can be obtained from
commercial sources such as Research Genetics (Huntsville, Ala.) and
the American Type Culture Collection (Manassas, Va.).
[0092] A library containing cDNA or genomic clones can be screened
with one or more polynucleotide probes based upon SEQ ID NO:1,
using standard methods (see, for example, Ausubel (1995) at pages
6-1 to 6-11).
[0093] Anti-zupar1 antibodies, produced as described below, can
also be used to isolate DNA sequences that encode human zupar1
genes from cDNA libraries. For example, the antibodies can be used
to screen .lambda.gt11 expression libraries, or the antibodies can
be used for immunoscreening following hybrid selection and
translation (see, for example, Ausubel (1995) at pages 6-12 to
6-16; Margolis et al., "Screening .lambda. expression libraries
with antibody and protein probes," in DNA Cloning 2: Expression
Systems, 2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford
University Press 1995)).
[0094] As an alternative, a zupar1 gene can be obtained by
synthesizing nucleic acid molecules using mutually priming long
oligonucleotides and the nucleotide sequences described herein
(see, for example, Ausubel (1995) at pages 8-8 to 8-9). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCR
Methods and Applications 2:266 (1993), Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.
4:299 (1995)).
[0095] The nucleic acid molecules of the present invention can also
be synthesized with "gene machines" using protocols such as the
phosphoramidite method. If chemically-synthesized double stranded
DNA is required for an application such as the synthesis of a gene
or a gene fragment, then each complementary strand is made
separately. The production of short genes (60 to 80 base pairs) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 base pairs), however, special
strategies may be required, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length. For reviews on polynucleotide synthesis,
see, for example, Glick and Pasternak, Molecular Biotechnology,
Principles and Applications of Recombinant DNA (ASM Press 1994),
Itakura et al., Annu. Rev. Biochem. 53:323 (1984), and Climie et
al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).
[0096] The sequence of a zupar1 cDNA or zupar1 genomic fragment can
be determined using standard methods. zupar1 polynucleotide
sequences disclosed herein can also be used as probes or primers to
clone 5' non-coding regions of a zupar1 gene. Promoter elements
from a zupar1 gene can be used to direct the expression of
heterologous genes in tissues of, for example, transgenic animals
or patients treated with gene therapy. The identification of
genomic fragments containing a zupar1 promoter or regulatory
element can be achieved using well-established techniques, such as
deletion analysis (see, generally, Ausubel (1995)).
[0097] Cloning of 5' flanking sequences also facilitates production
of zupar1 proteins by "gene activation," a technique disclosed in
U.S. Pat. No. 5,641,670. Briefly, expression of an endogenous
zupar1 gene in a cell is altered by introducing into the zupar1
locus a DNA construct comprising at least a targeting sequence, a
regulatory sequence, an exon, and an unpaired splice donor site.
The targeting sequence is a zupar1 5' non-coding sequence that
permits homologous recombination of the construct with the
endogenous zupar1 locus, whereby the sequences within the construct
become operably linked with the endogenous zupar1 coding sequence.
In this way, an endogenous zupar1 promoter can be replaced or
supplemented with other regulatory sequences to provide enhanced,
tissue-specific, or otherwise regulated expression.
[0098] 4. Production of zupar1 Gene Variants
[0099] The present invention provides a variety of nucleic acid
molecules, including DNA and RNA molecules, that encode the zupar1
polypeptides disclosed herein. Those skilled in the art will
readily recognize that, in view of the degeneracy of the genetic
code, considerable sequence variation is possible among these
polynucleotide molecules. SEQ ID NO:3 is a degenerate nucleotide
sequence that encompasses all nucleic acid molecules that encode
the zupar1 polypeptide of SEQ ID NO:2. Those skilled in the art
will recognize that the degenerate sequence of SEQ ID NO:3 also
provides all RNA sequences encoding SEQ ID NO:2, by substituting U
for T. Thus, the present invention contemplates zupar1
polypeptide-encoding nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, and their RNA equivalents.
[0100] Table 1 sets forth the one-letter codes used within SEQ ID
NO:3 to denote degenerate nucleotide positions. "Resolutions" are
the nucleotides denoted by a code letter. "Complement" indicates
the code for the complementary nucleotide(s). For example, the code
Y denotes either C or T, and its complement R denotes A or G, A
being complementary to T, and G being complementary to C.
1TABLE 1 Nucleotide Resolution Complement Resolution A A T T C C G
G G G C C T T A A R A.vertline.G Y C.vertline.T Y C.vertline.T R
A.vertline.G M A.vertline.C K G.vertline.T K G.vertline.T M
A.vertline.C S C.vertline.G S C.vertline.G W A.vertline.T W
A.vertline.T H A.vertline.C.vertline.T D A.vertline.G.vertline.T B
C.vertline.G.vertline.T V A.vertline.C.vertline.G V
A.vertline.C.vertline.G B C.vertline.G.vertline.T D
A.vertline.G.vertline.T H A.vertline.C.vertline.T N
A.vertline.C.vertline.G.vertline.T N
A.vertline.C.vertline.G.vertline.T
[0101] The degenerate codons used in SEQ ID NO:3, encompassing all
possible codons for a given amino acid, are set forth in Table
2.
2TABLE 2 One Amino Letter Degenerate Acid Code Codons Codon Cys C
TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT
ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA
GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG
GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG
CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L
CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT
TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR
Asn.vertline.Asp B RAY Glu.vertline.Gln Z SAR Any X NNN
[0102] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding an amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequence of SEQ ID NO:2.
Variant sequences can be readily tested for functionality as
described herein.
[0103] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev.
60:512 (1996). As used herein, the term "preferential codon usage"
or "preferential codons" is a term of art referring to protein
translation codons that are most frequently used in cells of a
certain species, thus favoring one or a few representatives of the
possible codons encoding each amino acid (see Table 2). For
example, the amino acid threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequence
disclosed in SEQ ID NO:3 serves as a template for optimizing
expression of polynucleotides in various cell types and species
commonly used in the art and disclosed herein. Sequences containing
preferential codons can be tested and optimized for expression in
various species, and tested for functionality as disclosed
herein.
[0104] The present invention further provides variant polypeptides
and nucleic acid molecules that represent counterparts from other
species (orthologs). These species include, but are not limited to
mammalian, avian, amphibian, reptile, fish, insect and other
vertebrate and invertebrate species. Of particular interest are
zupar1 polypeptides from mammalian species, including porcine,
ovine, rat, murine, bovine, canine, feline, equine, and other
primate polypeptides. Orthologs of zupar1 can be cloned using
information and compositions provided by the present invention in
combination with conventional cloning techniques. For example, a
cDNA can be cloned using mRNA obtained from a tissue or cell type
that expresses zupar1 as disclosed herein. Suitable sources of mRNA
can be identified by probing northern blots with probes designed
from the sequences disclosed herein. A library is then prepared
from mRNA of a positive tissue or cell line. A murine zupar1
homolog, as shown in SEQ ID NO:11, was identified from mouse testis
cDNA (Shibata et al., Genome Res. 10:1617-30, 2000).
[0105] A zupar1-encoding cDNA can then be isolated by a variety of
methods, such as by probing with a complete or partial cDNA or with
one or more sets of degenerate probes based on the disclosed
sequences. A cDNA can also be cloned using the polymerase chain
reaction with primers designed from the representative zupar1
sequences disclosed herein. Within an additional method, the cDNA
library can be used to transform or transfect host cells, and
expression of the cDNA of interest can be detected with an antibody
to zupar1 polypeptide. Similar techniques can also be applied to
the isolation of genomic clones.
[0106] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human
zupar1, and that allelic variation and alternative splicing are
expected to occur. Allelic variants of this sequence can be cloned
by probing cDNA or genomic libraries from different individuals
according to standard procedures. Allelic variants of the
nucleotide sequence shown in SEQ ID NO:1, including those
containing silent mutations and those in which mutations result in
amino acid sequence changes, are within the scope of the present
invention, as are proteins which are allelic variants of SEQ ID
NO:2. cDNA molecules generated from alternatively spliced mRNAs,
which retain the properties of the zupar1 polypeptide are included
within the scope of the present invention, as are polypeptides
encoded by such cDNAs and mRNAs. Allelic variants and splice
variants of these sequences can be cloned by probing cDNA or
genomic libraries from different individuals or tissues according
to standard procedures known in the art.
[0107] Within certain embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules comprising nucleotide sequences disclosed
herein. For example, such nucleic acid molecules can hybridize
under stringent conditions to nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, or to nucleic acid molecules
consisting of a nucleotide sequence complementary to SEQ ID NO:1.
In general, stringent conditions are selected to be about 5.degree.
C. lower than the thermal melting point (T.sub.m) for the specific
sequence at a defined ionic strength and pH. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of
the target sequence hybridizes to a perfectly matched probe.
[0108] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Stringent hybridization conditions encompass
temperatures of about 5-25.degree. C. below the T.sub.m of the
hybrid and a hybridization buffer having up to 1 M Na.sup.+. Higher
degrees of stringency at lower temperatures can be achieved with
the addition of formamide which reduces the T.sub.m of the hybrid
about 1.degree. C. for each 1% formamide in the buffer solution.
Generally, such stringent conditions include temperatures of
20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and 0-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and 0-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0109] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polypeptide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
which influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m are known in the art, and are specific for DNA, RNA and
DNA-RNA hybrids and polynucleotide probe sequences of varying
length (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989);
Ausubel et al., (eds.), Current Protocols in Molecular Biology
(John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide
to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and
Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence
analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and
Primer Premier 4.0 (Premier Biosoft International; Palo Alto,
Calif.), as well as sites on the Internet, are available tools for
analyzing a given sequence and calculating T.sub.m based on user
defined criteria. Such programs can also analyze a given sequence
under defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated T.sub.m. For smaller probes, <50 base
pairs, hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below. This allows for the maximum rate of
hybridization for DNA-DNA and DNA-RNA hybrids.
[0110] The length of the polynucleotide sequence influences the
rate and stability of hybrid formation. Smaller probe sequences,
<50 base pairs, reach equilibrium with complementary sequences
rapidly, but may form less stable hybrids. Incubation times of
anywhere from minutes to hours can be used to achieve hybrid
formation. Longer probe sequences come to equilibrium more slowly,
but form more stable complexes even at lower temperatures.
Incubations are allowed to proceed overnight or longer. Generally,
incubations are carried out for a period equal to three times the
calculated Cot time. Cot time, the time it takes for the
polynucleotide sequences to reassociate, can be calculated for a
particular sequence by methods known in the art.
[0111] The base pair composition of polynucleotide sequence will
effect the thermal stability of the hybrid complex, thereby
influencing the choice of hybridization temperature and the ionic
strength of the hybridization buffer. A-T pairs are less stable
than G-C pairs in aqueous solutions containing sodium chloride.
Therefore, the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence also
contribute positively to hybrid stability. In addition, the base
pair composition can be manipulated to alter the T.sub.m of a given
sequence. For example, 5-methyldeoxycytidine can be substituted for
deoxycytidine and 5-bromodeoxuridine can be substituted for
thymidine to increase the T.sub.m, whereas
7-deazz-2'-deoxyguanosine can be substituted for guanosine to
reduce dependence on T.sub.m.
[0112] The ionic concentration of the hybridization buffer also
affects the stability of the hybrid. Hybridization buffers
generally contain blocking agents such as Denhardt's solution
(Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA,
tRNA, milk powders (BLOTTO), heparin or SDS, and a Na.sup.+ source,
such as SSC (1.times.SSC: 0.15 M sodium chloride, 15 mM sodium
citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). By decreasing the ionic
concentration of the buffer, the stability of the hybrid is
increased. Typically, hybridization buffers contain from between 10
mM-1 M Na.sup.+. The addition of destabilizing or denaturing agents
such as formamide, tetralkylammonium salts, guanidinium cations or
thiocyanate cations to the hybridization solution will alter the
T.sub.m of a hybrid. Typically, formamide is used at a
concentration of up to 50% to allow incubations to be carried out
at more convenient and lower temperatures. Formamide also acts to
reduce non-specific background when using RNA probes.
[0113] As an illustration, a nucleic acid molecule encoding a
variant zupar1 polypeptide can be hybridized with a nucleic acid
molecule consisting of the nucleotide sequence of SEQ ID NO:1 (or
its complement) at 42.degree. C. overnight in a solution comprising
50% formamide, 5.times.SSC (1.times.SSC: 0.15 M sodium chloride and
15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6),
5.times.Denhardt's solution (100.times.Denhardt's solution: 2%
(w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v)
bovine serum albumin, 10% dextran sulfate, and 20 .mu.g/ml
denatured, sheared salmon sperm DNA. One of skill in the art can
devise variations of these hybridization conditions. For example,
the hybridization mixture can be incubated at a higher temperature,
such as about 65.degree. C., in a solution that does not contain
formamide. Moreover, premixed hybridization solutions are available
(e.g., EXPRESSHYB Hybridization Solution from CLONTECH
Laboratories, Inc.), and hybridization can be performed according
to the manufacturer's instructions.
[0114] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. For example, nucleic acid molecules encoding a
variant zupar1 polypeptide remain hybridized following stringent
washing conditions with a nucleic acid molecule consisting of the
nucleotide sequence of SEQ ID NO:1 (or its complement), in which
the wash stringency is equivalent to 0.5.times.-2.times.SSC with
0.1% SDS at 55-65.degree. C., including 0.5.times.SSC with 0.1% SDS
at 55.degree. C., or 2.times.SSC with 0.1% SDS at 65.degree. C. One
of skill in the art can readily devise equivalent conditions, for
example, by substituting the SSPE for SSC in the wash solution.
[0115] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. As an illustration, nucleic acid
molecules encoding a variant zupar1 polypeptide remain hybridized
following stringent washing conditions with a nucleic acid molecule
consisting of the nucleotide sequence of SEQ ID NO:1 (or its
complement), in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C.,
including 0.1.times.SSC with 0.1% SDS at 50.degree. C., or
0.2.times.SSC with 0.1% SDS at 65.degree. C.
[0116] The present invention also provides isolated zupar1
polypeptides that have a substantially similar sequence identity to
the polypeptide of SEQ ID NO:2, or orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides having 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the sequence shown in SEQ ID NO:2.
[0117] The present invention also contemplates zupar1 variant
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO:2, and a hybridization
assay, as described above. Such zupar1 variants include nucleic
acid molecules (1) that remain hybridized following stringent
washing conditions with a nucleic acid molecule consisting of the
nucleotide sequence of SEQ ID NO:1 (or its complement), in which
the wash stringency is equivalent to 0.5.times.-2.times.SSC with
0.1% SDS at 55-65.degree. C., and (2) that encode a polypeptide
having 70%, 80%, 90%, 95% 96%, 97%, 98% or 99% sequence identity to
the amino acid sequence of SEQ ID NO:2.
[0118] Alternatively, zupar1 variants can be characterized as
nucleic acid molecules (1) that remain hybridized following highly
stringent washing conditions with a nucleic acid molecule
consisting of the nucleotide sequence of SEQ ID NO:1 (or its
complement), in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C., and (2)
that encode a polypeptide having 70%, 80%, 90%, 95%, 96%, 97%, 98%
or 99% sequence identity to the amino acid sequence of SEQ ID
NO:2.
[0119] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA
89:10915 (1992). Briefly, two amino acid sequences are aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: ([Total number of identical matches]/[length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences])(100).
3 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0120] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative zupar1 variant. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990). Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:2) and a test sequence that have either the highest density of
identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the highest density
of identities are then re-scored by comparing the similarity of all
paired amino acids using an amino acid substitution matrix, and the
ends of the regions are "trimmed" to include only those residues
that contribute to the highest score. If there are several regions
with scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.
Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)),
which allows for amino acid insertions and deletions. Illustrative
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0121] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as described above.
[0122] The present invention includes nucleic acid molecules that
encode a polypeptide having a conservative amino acid change,
compared with the amino acid sequence of SEQ ID NO:2. That is,
variants can be obtained that contain one or more amino acid
substitutions of SEQ ID NO:2, in which an alkyl amino acid is
substituted for an alkyl amino acid in a zupar1 amino acid
sequence, an aromatic amino acid is substituted for an aromatic
amino acid in a zupar1 amino acid sequence, a sulfur-containing
amino acid is substituted for a sulfur-containing amino acid in a
zupar1 amino acid sequence, a hydroxy-containing amino acid is
substituted for a hydroxy-containing amino acid in a zupar1 amino
acid sequence, an acidic amino acid is substituted for an acidic
amino acid in a zupar1 amino acid sequence, a basic amino acid is
substituted for a basic amino acid in a zupar1 amino acid sequence,
or a dibasic monocarboxylic amino acid is substituted for a dibasic
monocarboxylic amino acid in a zupar1 amino acid sequence.
[0123] Among the common amino acids, for example, a "conservative
amino acid substitution" is illustrated by a substitution among
amino acids within each of the following groups: (1) glycine,
alanine, valine, leucine, and isoleucine, (2) phenylalanine,
tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate
and glutamate, (5) glutamine and asparagine, and (6) lysine,
arginine and histidine.
[0124] The BLOSUM62 table is an amino acid substitution matrix
derived from about 2,000 local multiple alignments of protein
sequence segments, representing highly conserved regions of more
than 500 groups of related proteins (Henikoff and Henikoff, Proc.
Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62
substitution frequencies can be used to define conservative amino
acid substitutions that may be introduced into the amino acid
sequences of the present invention. Although it is possible to
design amino acid substitutions based solely upon chemical
properties (as discussed above), the language "conservative amino
acid substitution" preferably refers to a substitution represented
by a BLOSUM62 value of greater than -1. For example, an amino acid
substitution is conservative if the substitution is characterized
by a BLOSUM62 value of 0, 1, 2, or 3. According to this system,
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
[0125] Particular variants of zupar1 are characterized by having
greater than 96%, at least 97%, at least 98%, or at least 99%
sequence identity to the corresponding amino acid sequence (e.g.,
the amino acid sequence of SEQ ID NO:2), wherein the variation in
amino acid sequence is due to one or more conservative amino acid
substitutions.
[0126] Conservative amino acid changes in a zupar1 gene can be
introduced by substituting nucleotides for the nucleotides recited
in SEQ ID NO:1. Such "conservative amino acid" variants can be
obtained, for example, by oligonucleotide-directed mutagenesis,
linker-scanning mutagenesis, mutagenesis using the polymerase chain
reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22;
and McPherson (ed.), Directed Mutagenesis: A Practical Approach
(IRL Press 1991)).
[0127] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is typically carried out in a
cell-free system comprising an E. coli S30 extract and commercially
available enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722 (1991), Ellman et al., Methods Enzymol. 202:301
(1991), Chung et al., Science 259:806 (1993), and Chung et al.,
Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0128] In a second method, translation is carried out in Xenopus
oocytes by microinjection of mutated mRNA and chemically
aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.
271:19991 (1996)). Within a third method, E. coli cells are
cultured in the absence of a natural amino acid that is to be
replaced (e.g., phenylalanine) and in the presence of the desired
non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).
The non-naturally occurring amino acid is incorporated into the
protein in place of its natural counterpart. See, Koide et al.,
Biochem. 33:7470 (1994). Naturally occurring amino acid residues
can be converted to non-naturally occurring species by in vitro
chemical modification. Chemical modification can be combined with
site-directed mutagenesis to further expand the range of
substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
[0129] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for zupar1 amino acid residues.
[0130] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Nat'l Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity as
disclosed below to identify amino acid residues that are critical
to the activity of the molecule. See also, Hilton et al., J. Biol.
Chem. 271:4699 (1996). Invariant amino acid residues include, the
cysteine residues corresponding to amino acid residues 23, 26, 34,
41, 47, 65, 71, 90, 91, and 95, and may include the Arg residue at
position 80 of SEQ ID NO:2. Mutations at the Arg 80 site may
modulate protease inhibition in a manner. By analogy, CD59 binds c8
or c9 of the complement membrane attack complex (MAC) which is
activated by thrombin. The CD59 active site may inhibit thrombin,
preventing the MAC from fully functioning and eliminating the site
reduces or eliminates the inhibitory effect and results in
inflammation.
[0131] The location of zupar1 receptor binding domains can also be
determined by physical analysis of structure, as determined by such
techniques as nuclear magnetic resonance, crystallography, electron
diffraction or photoaffinity labeling, in conjunction with mutation
of putative contact site amino acids. See, for example, de Vos et
al., Science 255:306 (1992), Smith et al., J. Mol. Biol. 224:899
(1992), and Wlodaver et al., FEBS Lett. 309:59 (1992). Moreover,
zupar1 labeled with biotin or FITC can be used for expression
cloning of zupar1 receptors.
[0132] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204, and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)).
[0133] Variants of the disclosed zupar1 nucleotide and polypeptide
sequences can also be generated through DNA shuffling as disclosed
by Stemmer, Nature 370:389 (1994), Stemmer, Proc. Nat'l Acad. Sci.
USA 91:10747 (1994), and international publication No. WO 97/20078.
Briefly, variant DNA molecules are generated by in vitro homologous
recombination by random fragmentation of a parent DNA followed by
reassembly using PCR, resulting in randomly introduced point
mutations. This technique can be modified by using a family of
parent DNA molecules, such as allelic variants or DNA molecules
from different species, to introduce additional variability into
the process. Selection or screening for the desired activity,
followed by additional iterations of mutagenesis and assay provides
for rapid "evolution" of sequences by selecting for desirable
mutations while simultaneously selecting against detrimental
changes.
[0134] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-zupar1 antibodies, can be
recovered from the host cells and rapidly sequenced using modem
equipment. These methods allow the rapid determination of the
importance of individual amino acid residues in a polypeptide of
interest, and can be applied to polypeptides of unknown
structure.
[0135] The present invention also includes "functional fragments"
of zupar1 polypeptides and nucleic acid molecules encoding such
functional fragments. Routine deletion analyses of nucleic acid
molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a zupar1 polypeptide. As an
illustration, DNA molecules having the nucleotide sequence of SEQ
ID NO:1 can be digested with Bal31 nuclease to obtain a series of
nested deletions. One alternative to exonuclease digestion is to
use oligonucleotide-directed mutagenesis to introduce deletions or
stop codons to specify production of a desired fragment.
Alternatively, particular fragments of a zupar1 gene can be
synthesized using the polymerase chain reaction.
[0136] As an illustration, studies on the truncation at either or
both termini of interferons have been summarized by Horisberger and
Di Marco, Pharmac. Ther. 66:507 (1995). Moreover, standard
techniques for functional analysis of proteins are described by,
for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993),
Content et al., "Expression and preliminary deletion analysis of
the 42 kDa 2-5A synthetase induced by human interferon," in
Biological Interferon Systems, Proceedings of ISIR-TNO Meeting on
Interferon Systems, Cantell (ed.), pages 65-72 (Nijhoff 1987),
Herschman, "The EGF Receptor," in Control of Animal Cell
Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0137] The present invention also contemplates functional fragments
of a zupar1 gene that has amino acid changes, compared with the
amino acid sequence of SEQ ID NO:2. A variant zupar1 gene can be
identified on the basis of structure by determining the level of
identity with nucleotide and amino acid sequences of SEQ ID NOs:1
and 2, as discussed above. An alternative approach to identifying a
variant gene on the basis of structure is to determine whether a
nucleic acid molecule encoding a potential variant zupar1 gene can
hybridize to a nucleic acid molecule having the nucleotide sequence
of SEQ ID NO:1, as discussed above.
[0138] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a zupar1
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0139] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein.
[0140] Antigenic epitope-bearing peptides and polypeptides
preferably contain at least four to ten amino acids, at least ten
to fifteen amino acids, or about 15 to about 30 amino acids of SEQ
ID NO:2. Such epitope-bearing peptides and polypeptides can be
produced by fragmenting a zupar1 polypeptide, or by chemical
peptide synthesis, as described herein. Moreover, epitopes can be
selected by phage display of random peptide libraries (see, for
example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and
Cortese et al., Curr. Opin. Biotechnol. 7:616 (1996)). Standard
methods for identifying epitopes and producing antibodies from
small peptides that comprise an epitope are described, for example,
by Mole, "Epitope Mapping," in Methods in Molecular Biology, Vol.
10, Manson (ed.), pages 105-116 (The Humana Press, Inc. 1992),
Price, "Production and Characterization of Synthetic
Peptide-Derived Antibodies," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 60-84 (Cambridge University Press 1995), and Coligan et al.
(eds.), Current Protocols in Immunology, pages 9.3.1 - 9.3.5 and
pages 9.4.1 - 9.4.11 (John Wiley & Sons 1997). Based on
structural analysis of zupar1 in multiple alignment with CD59,
predicted antigenic regions useful in generating blocking
antibodies should include, but not limited to a region centered on
residues E45, L66, R80, or T83. Bodian et al., Exp. Med.
185:507-16, 1997. The regions might include, but are not limited to
amino acid residues 40-55, 59-75, 73-89, or 76-90 of SEQ ID
NO:2.
[0141] For any zupar1 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above. Moreover,
those of skill in the art can use standard software to devise
zupar1 variants based upon the nucleotide and amino acid sequences
described herein. Accordingly, the present invention includes a
computer-readable medium encoded with a data structure that
provides at least one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
Suitable forms of computer-readable media include magnetic media
and optically-readable media. Examples of magnetic media include a
hard or fixed drive, a random access memory (RAM) chip, a floppy
disk, digital linear tape (DLT), a disk cache, and a ZIP disk.
Optically readable media are exemplified by compact discs (e.g.,
CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable),
and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM,
and DVD+RW).
[0142] 5. Production of zupar1 Fusion Proteins
[0143] Fusion proteins of zupar1 can be used to express zupar1 in a
recombinant host, and to isolate expressed zupar1. One type of
fusion protein comprises a peptide that guides a zupar1 polypeptide
from a recombinant host cell. To direct a zupar1 polypeptide into
the secretory pathway of a eukaryotic host cell, a secretory signal
sequence (also known as a signal peptide, a leader sequence, prepro
sequence or pre sequence) is provided in the zupar1 expression
vector. While the secretory signal sequence may be derived from
zupar1, a suitable signal sequence may also be derived from another
secreted protein or synthesized de novo. The secretory signal
sequence is operably linked to a zupar1-encoding sequence such that
the two sequences are joined in the correct reading frame and
positioned to direct the newly synthesized polypeptide into the
secretory pathway of the host cell. Secretory signal sequences are
commonly positioned 5' to the nucleotide sequence encoding the
polypeptide of interest, although certain secretory signal
sequences may be positioned elsewhere in the nucleotide sequence of
interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland
et al., U.S. Pat. No. 5,143,830).
[0144] While the secretory signal sequence a protein produced by
mammalian cells (e.g., tissue-type plasminogen activator signal
sequence, as described, for example, in U.S. Pat. No. 5,641,655) is
useful for expression of zupar1 in recombinant mammalian hosts, a
yeast signal sequence is preferred for expression in yeast cells.
Examples of suitable yeast signal sequences are those derived from
yeast mating phermone .alpha.-factor (encoded by the MF.alpha.1
gene), invertase (encoded by the SUC2 gene), or acid phosphatase
(encoded by the PHO5 gene). See, for example, Romanos et al.,
"Expression of Cloned Genes in Yeast," in DNA Cloning 2: A
Practical Approach, 2.sup.nd Edition, Glover and Hames (eds.),
pages 123-167 (Oxford University Press 1995).
[0145] In bacterial cells, it is often desirable to express a
heterologous protein as a fusion protein to decrease toxicity,
increase stability, and to enhance recovery of the expressed
protein. For example, zupar1 can be expressed as a fusion protein
comprising a glutathione S-transferase polypeptide. Glutathione
S-transferease fusion proteins are typically soluble, and easily
purifiable from E. coli lysates on immobilized glutathione columns.
In similar approaches, a zupar1 fusion protein comprising a maltose
binding protein polypeptide can be isolated with an amylose resin
column, while a fusion protein comprising the C-terminal end of a
truncated Protein A gene can be purified using IgG-Sepharose.
Established techniques for expressing a heterologous polypeptide as
a fusion protein in a bacterial cell are described, for example, by
Williams et al., "Expression of Foreign Proteins in E. coli Using
Plasmid Vectors and Purification of Specific Polyclonal
Antibodies," in DNA Cloning 2: A Practical Approach, 2.sup.nd
Edition, Glover and Hames (Eds.), pages 15-58 (Oxford University
Press 1995). In addition, commercially available expression systems
are available. For example, the PINPOINT Xa protein purification
system (Promega Corporation; Madison, Wis.) provides a method for
isolating a fusion protein comprising a polypeptide that becomes
biotinylated during expression with a resin that comprises
avidin.
[0146] Peptide tags that are useful for isolating heterologous
polypeptides expressed by either prokaryotic or eukaryotic cells
include polyHistidine tags (which have an affinity for
nickel-chelating resin), c-myc tags, calmodulin binding protein
(isolated with calmodulin affinity chromatography), substance P,
the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu
tag, and the FLAG tag (which binds with anti-FLAG antibodies). See,
for example, Luo et al., Arch. Biochem. Biophys. 329:215 (1996),
Morganti et al., Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng
et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide tags are available, for example, from Sigma-Aldrich
Corporation (St. Louis, Mo.).
[0147] Another form of fusion protein comprises a zupar1
polypeptide and an immunoglobulin heavy chain constant region,
typically an F.sub.C fragment, which contains two constant region
domains and a hinge region but lacks the variable region. As an
illustration, Chang et al., U.S. Pat. No. 5,723,125, describe a
fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment, in which the C-terminal of the
interferon is linked to the N-terminal of the Fc fragment by a
peptide linker moiety. An example of a peptide linker is a peptide
comprising primarily a T cell inert sequence, which is
immunologically inert. An exemplary peptide linker has the amino
acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO:4). In such a fusion
protein, an illustrative Fc moiety is a human .gamma.4 chain, which
is stable in solution and has little or no complement activating
activity. Accordingly, the present invention contemplates a zupar1
fusion protein that comprises a zupar1 moiety and a human Fc
fragment, wherein the C-terminus of the zupar1 moiety is attached
to the N-terminus of the Fc fragment via a peptide linker, such as
a peptide consisting of the amino acid sequence of SEQ ID NO:4. The
zupar1 moiety can be a zupar1 molecule or a fragment thereof. Such
fragments can include, but are not limited to the polypeptides of
SEQ ID NO:5-10.
[0148] In another variation, a zupar1 fusion protein comprises an
IgG sequence, a zupar1 moiety covalently joined to the amino
terminal end of the IgG sequence, and a signal peptide that is
covalently joined to the amino terminal of the zupar1 moiety,
wherein the IgG sequence consists of the following elements in the
following order: a hinge region, a CH.sub.2 domain, and a CH.sub.3
domain. Accordingly, the IgG sequence lacks a CH.sub.1 domain. The
zupar1 moiety displays a zupar1 activity, as described herein, such
as the ability to bind with a zupar1 antibody. This general
approach to producing fusion proteins that comprise both antibody
and non-antibody portions has been described by LaRochelle et al.,
EP 742830 (WO 95/21258).
[0149] Fusion proteins comprising a zupar1 moiety and an Fc moiety
can be used, for example, as an in vitro assay tool. For example,
the presence of a zupar1 receptor in a biological sample can be
detected using a zupar1-antibody fusion protein, in which the
zupar1 moiety is used to target the cognate receptor, and a
macromolecule, such as Protein A or anti-Fc antibody, is used to
detect the bound fusion protein-receptor complex. Furthermore, such
fusion proteins can be used to identify agonists and antagonists
that interfere with the binding of zupar1 to its receptor.
[0150] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating the components. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. General methods for
enzymatic and chemical cleavage of fusion proteins are described,
for example, by Ausubel (1995) at pages 16-19 to 16-25.
[0151] 6. Production of zupar1 Polypeptides in Cultured Cells
[0152] The polypeptides of the present invention, including
full-length polypeptides, functional fragments, and fusion
proteins, can be produced in recombinant host cells following
conventional techniques. To express a zupar1 gene, a nucleic acid
molecule encoding the polypeptide must be operably linked to
regulatory sequences that control transcriptional expression in an
expression vector and then, introduced into a host cell. In
addition to transcriptional regulatory sequences, such as promoters
and enhancers, expression vectors can include translational
regulatory sequences and a marker gene which is suitable for
selection of cells that carry the expression vector.
[0153] Expression vectors that are suitable for production of a
foreign protein in eukaryotic cells typically contain (1)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector in a bacterial host; (2)
eukaryotic DNA elements that control initiation of transcription,
such as a promoter; and (3) DNA elements that control the
processing of transcripts, such as a transcription
termination/polyadenylation sequence. As discussed above,
expression vectors can also include nucleotide sequences encoding a
secretory sequence that directs the heterologous polypeptide into
the secretory pathway of a host cell. For example, a zupar1
expression vector may comprise a zupar1 gene and a secretory
sequence derived from a zupar1 gene or another secreted gene.
[0154] zupar1 proteins of the present invention may be expressed in
mammalian cells. Examples of suitable mammalian host cells include
African green monkey kidney cells (Vero; ATCC CRL 1587), human
embryonic kidney cells (293-HEK; ATCC CRL 1573), baby hamster
kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314),
canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary
cells (CHO-K1; ATCC CCL61; CHO DG44 (Chasin et al., Som. Cell.
Molec. Genet. 12:555, 1986)), rat pituitary cells (GH1; ATCC
CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;
ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).
[0155] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, simian virus, or the like, in
which the regulatory signals are associated with a particular gene
which has a high level of expression. Suitable transcriptional and
translational regulatory sequences also can be obtained from
mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0156] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273
(1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304
(1981)), the Rous sarcoma virus promoter (Gorman et al., Proc.
Nat'l Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter
(Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering: Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0157] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
zupar1 gene expression in mammalian cells if the prokaryotic
promoter is regulated by a eukaryotic promoter (Zhou et al., Mol.
Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl. Acids Res.
19:4485 (1991)).
[0158] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
Preferably, the transfected cells are selected and propagated to
provide recombinant host cells that comprise the expression vector
stably integrated in the host cell genome. Techniques for
introducing vectors into eukaryotic cells and techniques for
selecting such stable transformants using a dominant selectable
marker are described, for example, by Ausubel (1995) and by Murray
(ed.), Gene Transfer and Expression Protocols (Humana Press
1991).
[0159] For example, one suitable selectable marker is a gene that
provides resistance to the antibiotic neomycin. In this case,
selection is carried out in the presence of a neomycin-type drug,
such as G-418 or the like. Selection systems can also be used to
increase the expression level of the gene of interest, a process
referred to as "amplification." Amplification is carried out by
culturing transfectants in the presence of a low level of the
selective agent and then increasing the amount of selective agent
to select for cells that produce high levels of the products of the
introduced genes. An exemplary amplifiable selectable marker is
dihydrofolate reductase, which confers resistance to methotrexate.
Other drug resistance genes (e.g., hygromycin resistance,
multi-drug resistance, puromycin acetyltransferase) can also be
used. Alternatively, markers that introduce an altered phenotype,
such as green fluorescent protein, or cell surface proteins (e.g.,
CD4, CD8, Class I MHC, and placental alkaline phosphatase) may be
used to sort transfected cells from untransfected cells by such
means as FACS sorting or magnetic bead separation technology.
[0160] zupar1 polypeptides can also be produced by cultured cells
using a viral delivery system. Exemplary viruses for this purpose
include adenovirus, herpesvirus, vaccinia virus and
adeno-associated virus (AAV). Adenovirus, a double-stranded DNA
virus, is currently the best studied gene transfer vector for
delivery of heterologous nucleic acid (for a review, see Becker et
al., Meth. Cell Biol. 43:161 (1994), and Douglas and Curiel,
Science & Medicine 4:44 (1997)). Advantages of the adenovirus
system include the accommodation of relatively large DNA inserts,
the ability to grow to high-titer, the ability to infect a broad
range of mammalian cell types, and flexibility that allows use with
a large number of available vectors containing different
promoters.
[0161] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. An
option is to delete the essential E1 gene from the viral vector,
which results in the inability to replicate unless the E1 gene is
provided by the host cell. For example, adenovirus vector infected
human 293 cells (ATCC Nos. CRL-1573, 45504, 45505) can be grown as
adherent cells or in suspension culture at relatively high cell
density to produce significant amounts of protein (see Garnier et
al., Cytotechnol. 15:145 (1994)).
[0162] zupar1 genes may also be expressed in other higher
eukaryotic cells, such as avian, fungal, insect, yeast, or plant
cells. The baculovirus system provides an efficient means to
introduce cloned zupar1 genes into insect cells. Suitable
expression vectors are based upon the Autographa californica
multiple nuclear polyhedrosis virus (AcMNPV), and contain
well-known promoters such as Drosophila heat shock protein (hsp) 70
promoter, Autographa californica nuclear polyhedrosis virus
immediate-early gene promoter (ie-1) and the delayed early 39K
promoter, baculovirus p10 promoter, and the Drosophila
metallothionein promoter. A second method of making recombinant
baculovirus utilizes a transposon-based system described by Luckow
(Luckow, et al., J. Virol. 67:4566 (1993)). This system, which
utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life
Technologies, Rockville, Md.). This system utilizes a transfer
vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to
move the DNA encoding the zupar1 polypeptide into a baculovirus
genome maintained in E. coli as a large plasmid called a "bacmid."
See, Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990),
Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, and
Rapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer
vectors can include an in-frame fusion with DNA encoding an epitope
tag at the C- or N-terminus of the expressed zupar1 polypeptide,
for example, a Glu-Glu epitope tag (Grussenmeyer et al., Proc.
Nat'l Acad. Sci. 82:7952 (1985)). Using a technique known in the
art, a transfer vector containing a zupar1 gene is transformed into
E. coli, and screened for bacmids which contain an interrupted lacZ
gene indicative of recombinant baculovirus. The bacmid DNA
containing the recombinant baculovirus genome is then isolated
using common techniques.
[0163] The illustrative PFASTBAC vector can be modified to a
considerable degree. For example, the polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins (see, for example,
Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et
al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J.
Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a
short or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native zupar1 secretory signal sequences with secretory signal
sequences derived from insect proteins. For example, a secretory
signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), or
baculovirus gp67 (PharMingen: San Diego, Calif.) can be used in
constructs to replace the native zupar1 secretory signal
sequence.
[0164] The recombinant virus or bacmid is used to transfect host
cells. Suitable insect host cells include cell lines derived from
IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such
as Sf9 (ATCC CRL 1711), Sf2AE, and Sf21 (Invitrogen Corporation;
San Diego, Calif.), as well as Drosophila Schneider-2 cells, and
the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
can be used to grow and to maintain the cells. Suitable media are
Sf900 II.TM. (Life Technologies) or ESF 921.TM. (Expression
Systems) for the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences,
Lenexa, Kans.) or Express FiveO.TM. (Life Technologies) for the T.
ni cells. When recombinant virus is used, the cells are typically
grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3.
[0165] Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus Vectors," in Methods in Molecular Biology, Volume 7:
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168
(The Humana Press, Inc. 1991), by Patel et al., "The baculovirus
expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University
Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by
Richardson (ed.), Baculovirus Expression Protocols (The Humana
Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0166] Fungal cells, including yeast cells, can also be used to
express the genes described herein. Yeast species of particular
interest in this regard include Saccharomyces cerevisiae, Pichia
pastoris, and Pichia methanolica. Suitable promoters for expression
in yeast include promoters from GAL1 (galactose), PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
Many yeast cloning vectors have been designed and are readily
available. These vectors include YIp-based vectors, such as YIp5,
YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCp
vectors, such as YCp19. Methods for transforming S. cerevisiae
cells with exogenous DNA and producing recombinant polypeptides
therefrom are disclosed by, for example, Kawasaki, U.S. Pat. No.
4,599,311, Kawasaki et al., U.S. Pat. No. 4,931,373, Brake, U.S.
Pat. No. 4,870,008, Welch et al., U.S. Pat. No. 5,037,743, and
Murray et al., U.S. Pat. No. 4,845,075. Transformed cells are
selected by phenotype determined by the selectable marker, commonly
drug resistance or the ability to grow in the absence of a
particular nutrient (e.g., leucine). An illustrative vector system
for use in Saccharomyces cerevisiae is the POT1 vector system
disclosed by Kawasaki et al. (U.S. Pat. No. 4,931,373), which
allows transformed cells to be selected by growth in
glucose-containing media. Additional suitable promoters and
terminators for use in yeast include those from glycolytic enzyme
genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman et
al., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092)
and alcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,
5,063,154, 5,139,936, and 4,661,454.
[0167] Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459 (1986), and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0168] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA
molecules for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which are
preferably linearized prior to transformation. For polypeptide
production in P. methanolica, it is preferred that the promoter and
terminator in the plasmid be that of a P. methanolica gene, such as
a P. methanolica alcohol utilization gene (AUG1 or AUG2). Other
useful promoters include those of the dihydroxyacetone synthase
(DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. An illustrative
selectable marker for use in Pichia methanolica is a P. methanolica
ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole
carboxylase (AIRC; EC 4.1.1.21), and which allows ade2 host cells
to grow in the absence of adenine. For large-scale, industrial
processes where it is desirable to minimize the use of methanol, it
is preferred to use host cells in which both methanol utilization
genes (AUG1 and AUG2) are deleted. For production of secreted
proteins, host cells deficient in vacuolar protease genes (PEP4 and
PRB1) are preferred. Electroporation is used to facilitate the
introduction of a plasmid containing DNA encoding a polypeptide of
interest into P. methanolica cells. P. methanolica cells can be
transformed by electroporation using an exponentially decaying,
pulsed electric field having a field strength of from 2.5 to 4.5
kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from
1 to 40 milliseconds, most preferably about 20 milliseconds.
[0169] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. Methods
for introducing expression vectors into plant tissue include the
direct infection or co-cultivation of plant tissue with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA
injection, electroporation, and the like. See, for example, Horsch
et al., Science 227:1229 (1985), Klein et al., Biotechnology 10:268
(1992), and Miki et al, "Procedures for Introducing Foreign DNA
into Plants," in Methods in Plant Molecular Biology and
Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,
1993).
[0170] Alternatively, zupar1 genes can be expressed in prokaryotic
host cells. Suitable promoters that can be used to express zupar1
polypeptides in a prokaryotic host are well-known to those of skill
in the art and include promoters capable of recognizing the T4, T3,
Sp6 and T7 polymerases, the P.sub.R and P.sub.L promoters of
bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac,
lpp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.
subtilis, the promoters of the bacteriophages of Bacillus,
Streptomyces promoters, the int promoter of bacteriophage lambda,
the bla promoter of pBR322, and the CAT promoter of the
chloramphenicol acetyl transferase gene. Prokaryotic promoters have
been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et
al., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins
1987), and by Ausubel et al. (1995).
[0171] Useful prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF',
DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL
Press 1985)).
[0172] When expressing a zupar1 polypeptide in bacteria such as E.
coli, the polypeptide may be retained in the cytoplasm, typically
as insoluble granules, or may be directed to the periplasmic space
by a bacterial secretion sequence. In the former case, the cells
are lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0173] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et aL, "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), page 101 (John Wiley & Sons, Inc. 1996)).
[0174] Standard methods for introducing expression vectors into
bacterial, yeast, insect, and plant cells are provided, for
example, by Ausubel (1995).
[0175] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc. 1995).
[0176] As an alternative, polypeptides of the present invention can
be synthesized by exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill
in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd
Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept.
Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach (IRL Press 1989), Fields and Colowick,
"Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical
Approaches to the Synthesis of Peptides and Proteins (CRC Press,
Inc. 1997)). Variations in total chemical synthesis strategies,
such as "native chemical ligation" and "expressed protein ligation"
are also standard (see, for example, Dawson et al., Science 266:776
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997),
Dawson, Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l
Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol.
Chem. 273:16205 (1998)).
[0177] 7. Isolation of zupar1 Polypeptides
[0178] The polypeptides of the present invention can be purified to
at least about 80% purity, to at least about 90% purity, to at
least about 95% purity, or greater than 95% purity with respect to
contaminating macromolecules, particularly other proteins and
nucleic acids, and free of infectious and pyrogenic agents. The
polypeptides of the present invention may also be purified to a
pharmaceutically pure state, which is greater than 99.9% pure.
Certain purified polypeptide preparations are substantially free of
other polypeptides, particularly other polypeptides of animal
origin.
[0179] Fractionation and/or conventional purification methods can
be used to obtain preparations of zupar1 purified from natural
sources, and recombinant zupar1 polypeptides and fusion zupar1
polypeptides purified from recombinant host cells. In general,
ammonium sulfate precipitation and acid or chaotrope extraction may
be used for fractionation of samples. Exemplary purification steps
may include hydroxyapatite, size exclusion, FPLC and reverse-phase
high performance liquid chromatography. Suitable chromatographic
media include derivatized dextrans, agarose, cellulose,
polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and
Q derivatives are preferred. Exemplary chromatographic media
include those media derivatized with phenyl, butyl, or octyl
groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl
650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia)
and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso
Haas) and the like. Suitable solid supports include glass beads,
silica-based resins, cellulosic resins, agarose beads, cross-linked
agarose beads, polystyrene beads, cross-linked polyacrylamide
resins and the like that are insoluble under the conditions in
which they are to be used. These supports may be modified with
reactive groups that allow attachment of proteins by amino groups,
carboxyl groups, sulfhydryl groups, hydroxyl groups and/or
carbohydrate moieties.
[0180] Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Selection of a particular
method for polypeptide isolation and purification is a matter of
routine design and is determined in part by the properties of the
chosen support. See, for example, Affinity Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
[0181] Additional variations in zupar1 isolation and purification
can be devised by those of skill in the art. For example,
anti-zupar1 antibodies, obtained as described below, can be used to
isolate large quantities of protein by immunoaffinity purification.
Moreover, methods for binding ligands, such as zupar1, to receptor
polypeptides bound to support media are well known in the art.
[0182] The polypeptides of the present invention can also be
isolated by exploitation of particular properties. For example,
immobilized metal ion adsorption (IMAC) chromatography can be used
to purify histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1
(1985)). Histidine-rich proteins will be adsorbed to this matrix
with differing affinities, depending upon the metal ion used, and
will be eluted by competitive elution, lowering the pH, or use of
strong chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (M. Deutscher,
(ed.), Meth. Enzymol. 182:529 (1990)). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification.
[0183] zupar1 polypeptides or fragments thereof may also be
prepared through chemical synthesis, as described above. zupar1
polypeptides or may not include an initial methionine amino acid
residue, may be glycosylated or non-glycosylated, and may be
monomers or multimers.
[0184] The present invention also contemplates chemically modified
zupar1 compositions, in which a zupar1 polypeptide is linked with a
polymer. Typically, the polymer is water soluble so that the zupar1
conjugate does not precipitate in an aqueous environment, such as a
physiological environment. An example of a suitable polymer is one
that has been modified to have a single reactive group, such as an
active ester for acylation, or an aldehyde for alkylation, In this
way, the degree of polymerization can be controlled. An example of
a reactive aldehyde is polyethylene glycol propionaldehyde, or
mono-(C.sub.1-C.sub.10) alkoxy, or aryloxy derivatives thereof
(see, for example, Harris, et al., U.S. Pat. No. 5,252,714). The
polymer may be branched or unbranched. Moreover, a mixture of
polymers can be used to produce zupar1 conjugates.
[0185] For example, zupar1 conjugates can comprise pharmaceutically
acceptable water-soluble polymer moieties. Suitable water-soluble
polymers include polyethylene glycol (PEG), monomethoxy-PEG,
mono-(C.sub.1-C.sub.10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinyl
pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,
bis-succinimidyl carbonate PEG, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or
other carbohydrate-based polymers. Suitable PEG may have a
molecular weight from about 600 to about 60,000, including, for
example, 5,000, 12,000, 20,000 and 25,000. A zupar1 conjugate can
also comprise a mixture of such water-soluble polymers. Anti-zupar1
antibodies or anti-idiotype antibodies can also be conjugated with
a water-soluble polymer.
[0186] The present invention contemplates compositions comprising a
peptide or polypeptide described herein. Such compositions can
further comprise a carrier. The carrier can be a conventional
organic or inorganic carrier. Examples of carriers include water,
buffer solution, alcohol, propylene glycol, macrogol, sesame oil,
corn oil, and the like.
[0187] Peptides and polypeptides of the present invention comprise
at least six, preferably at least nine, and more preferably at
least 15 contiguous amino acid residues of SEQ ID NO:2. Within
certain embodiments of the invention, the polypeptides comprise 20,
30, 40, or more contiguous residues of these amino acid sequences.
These polypeptide fragments can include, but are not limited to,
the polypeptides of SEQ ID NOs:5-10. Nucleic acid molecules
encoding such peptides and polypeptides are useful as polymerase
chain reaction primers and probes.
[0188] 8. Production of Antibodies to zupar1 Proteins
[0189] Antibodies to zupar1 can be obtained, for example, using as
an antigen the product of a zupar1 expression vector or zupar1
isolated from a natural source. Particularly useful anti-zupar1
antibodies "bind specifically" with zupar1. Antibodies are
considered to be specifically binding if the antibodies exhibit at
least one of the following two properties: (1) antibodies bind to
zupar1 with a threshold level of binding activity, and (2)
antibodies do not significantly cross-react with polypeptides
related to zupar1, such as urokinase plasminogen activators, CD59,
and vasopressin-neurophysin precursors.
[0190] With regard to the first characteristic, antibodies
specifically bind if they bind to a zupar1 polypeptide, peptide or
epitope with a binding affinity (K.sub.a) of 10.sup.6 M.sup.-1 or
greater, preferably 10.sup.7 M.sup.-1 or greater, more preferably
10.sup.8 M.sup.-1 or greater, and most preferably 10.sup.9 M.sup.-1
or greater. The binding affinity of an antibody can be readily
determined by one of ordinary skill in the art, for example, by
Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)).
With regard to the second characteristic, antibodies do not
significantly cross-react with related polypeptide molecules, for
example, if they detect zupar1, but not known polypeptides (e.g.,
GPI-anchored proteins) using a standard Western blot analysis.
[0191] Anti-zupar1 antibodies can be produced using antigenic
zupar1 epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, preferably between 15 to about
30 amino acids contained within SEQ ID NO:2. However, peptides or
polypeptides comprising a larger portion of an amino acid sequence
of the invention, containing from 30 to 50 amino acids, or any
length up to and including the entire amino acid sequence of a
polypeptide of the invention, also are useful for inducing
antibodies that bind with zupar1. Such antigenic regions include,
but are not limited to a region centered on residues E45, L66, RSO,
or T83. The regions might include, but are not limited to amino
acid residues 40-55, 59-75, 73-89, or 76-90 of SEQ ID NO:2.
[0192] It is desirable that the amino acid sequence of the
epitope-bearing peptide is selected to provide substantial
solubility in aqueous solvents (i.e., the sequence includes
relatively hydrophilic residues, while hydrophobic residues are
preferably avoided). Moreover, amino acid sequences containing
proline residues may be also be desirable for antibody
production.
[0193] Potential antigenic sites in zupar1 may be identified using
the Jameson-Wolf method, Jameson and Wolf, CABIOS 4:181, (1988), as
implemented by the PROTEAN program (version 3.14) of LASERGENE
(DNASTAR; Madison, Wis.). Default parameters were used in this
analysis.
[0194] The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for protein
structural prediction. Briefly, the Hopp-Woods method, Hopp et al.,
Proc. Nat'l Acad. Sci. USA 78:3824 (1981), can be used to identify
amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). The Emini's
method, Emini et al., J. Virology 55:836 (1985), can then be used
to calculate surface probabilities (parameter: surface decision
threshold (0.6)=1). The Karplus-Schultz method, Karplus and
Schultz, Naturwissenschaften 72:212 (1985), can be used to predict
backbone chain flexibility (parameter: flexibility threshold
(0.2)=1). These methods may be followed by application of secondary
structure predictions to the data using the methods of Chou-Fasman,
Chou, "Prediction of Protein Structural Classes from Amino Acid
Composition," in Prediction of Protein Structure and the Principles
of Protein Conformation, Fasman (ed.), pages 549-586 (Plenum Press
1990), and Garnier-Robson, Garnier et al., J. Mol. Biol. 120:97
(1978) (Chou-Fasman parameters: conformation table=64 proteins;
.alpha. region threshold=103; .beta. region threshold=105;
Garnier-Robson parameters: .alpha. and .beta. decision
constants=0). Flexibility parameters and hydropathy/solvent
accessibility factors may be combined to determine a surface
contour value, designated as the "antigenic index." Finally, a peak
broadening function may be applied to the antigenic index, which
broadens major surface peaks by adding 20, 40, 60, or 80% of the
respective peak value to account for additional free energy derived
from the mobility of surface regions relative to interior
regions.
[0195] Polyclonal antibodies to recombinant zupar1 protein or to
zupar1 isolated from natural sources can be prepared using methods
well-known to those of skill in the art. Antibodies can also be
generated using a zupar1-glutathione transferase fusion protein,
which is similar to a method described by Burrus and McMahon, Exp.
Cell. Res. 220:363 (1995). General methods for producing polyclonal
antibodies are described, for example, by Green et al., "Production
of Polyclonal Antisera," in Immunochemical Protocols (Manson, ed.),
pages 1-5 (Humana Press 1992), and Williams et al., "Expression of
foreign proteins in E. coli using plasmid vectors and purification
of specific polyclonal antibodies," in DNA Cloning 2: Expression
Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford
University Press 1995).
[0196] The immunogenicity of a zupar1 polypeptide can be increased
through the use of an adjuvant, such as alum (aluminum hydroxide)
or Freund's complete or incomplete adjuvant. Polypeptides useful
for immunization also include fusion polypeptides, such as fusions
of zupar1 or a portion thereof with an immunoglobulin polypeptide
or with maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like," such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0197] Although polyclonal antibodies are typically raised in
animals such as horse, cow, dog, chicken, rat, mouse, rabbit, goat,
guinea pig, or sheep, an anti-zupar1 antibody of the present
invention may also be derived from a subhuman primate antibody.
General techniques for raising diagnostically and therapeutically
useful antibodies in baboons may be found, for example, in
Goldenberg et al., international patent publication No. WO
91/11465, and in Losman et al., Int. J. Cancer 46:310 (1990).
[0198] Alternatively, monoclonal anti-zupar1 antibodies can be
generated. Rodent monoclonal antibodies to specific antigens may be
obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0199] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a zupar1 gene product, verifying
the presence of antibody production by removing a serum sample,
removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0200] In addition, an anti-zupar1 antibody of the present
invention may be derived from a human monoclonal antibody. Human
monoclonal antibodies are obtained from transgenic mice that have
been engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, for example, by Green et al., Nature Genet.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et
al., Int. Immun. 6:579 (1994).
[0201] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0202] For particular uses, it may be desirable to prepare
fragments of anti-zupar1 antibodies. Such antibody fragments can be
obtained, for example, by proteolytic hydrolysis of the antibody.
Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies by conventional methods. As an illustration,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al.,
in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967),
and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0203] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0204] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437 (1992)).
[0205] The Fv fragments may comprise V.sub.H and V.sub.L chains
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science
242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et
al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0206] As an illustration, an scFV can be obtained by exposing
lymphocytes to zupar1 polypeptide in vitro, and selecting antibody
display libraries in phage or similar vectors (for instance,
through use of immobilized or labeled zupar1 protein or peptide).
Genes encoding polypeptides having potential zupar1 polypeptide
binding domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides which interact with a
known target which can be a protein or polypeptide, such as a
ligand or receptor, a biological or synthetic macromolecule, or
organic or inorganic substances. Techniques for creating and
screening such random peptide display libraries are known in the
art (Ladner et al., U.S. Pat. No. 5,223,409, Ladner et al., U.S.
Pat. No. 4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner
et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of
Peptides and Proteins (Academic Press, Inc. 1996)) and random
peptide display libraries and kits for screening such libraries are
available commercially, for instance from CLONTECH Laboratories,
Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New
England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB
Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the zupar1 sequences disclosed
herein to identify proteins which bind to zupar1.
[0207] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0208] Alternatively, an anti-zupar1 antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer
et al., J. Immun. 150:2844 (1993), Sudhir (ed.), Antibody
Engineering Protocols (Humana Press, Inc. 1995), Kelley,
"Engineering Therapeutic Antibodies," in Protein Engineering:
Principles and Practice, Cleland et al. (eds.), pages 399-434 (John
Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat. No.
5,693,762 (1997).
[0209] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-zupar1 antibodies or antibody
fragments, using standard techniques. See, for example, Green et
al., "Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,
monoclonal anti-idiotype antibodies can be prepared using
anti-zupar1 antibodies or antibody fragments as immunogens with the
techniques, described above. As another alternative, humanized
anti-idiotype antibodies or subhuman primate anti-idiotype
antibodies can be prepared using the above-described techniques.
Methods for producing anti-idiotype antibodies are described, for
example, by Irie, U.S. Pat. No. 5,208,146, Greene, et. al., U.S.
Pat. No. 5,637,677, and Varthakavi and Minocha, J. Gen. Virol.
77:1875 (1996).
[0210] Anti-idiotype zupar1 antibodies, as well as zupar1
polypeptides can be used to identify and to isolate zupar1
receptors. For example, proteins and peptides of the present
invention can be immobilized on a column and used to bind receptor
proteins from membrane preparations that are run over the column
(Hermanson et al. (eds.), Immobilized Affinity Ligand Techniques,
pages 195-202 (Academic Press 1992)). Radiolabeled or affinity
labeled zupar1 polypeptides can also be used to identify or to
localize zupar1 receptors in a biological sample (see, for example,
Deutscher (ed.), Methods in Enzymol., vol. 182, pages 721-37
(Academic Press 1990); Brunner et al., Ann. Rev. Biochem. 62:483
(1993); Fedan et al., Biochem. Pharmacol. 33:1167 (1984)). Also
see, Varthakavi and Minocha, J. Gen. Virol. 77:1875 (1996), who
describe the use of anti-idiotype antibodies for receptor
identification.
[0211] 9. Zupar1 Polypeptides and Analogs
[0212] One general class of zupar1 analogs are zupar1 variants
having an amino acid sequence that is a mutation of the amino acid
sequence disclosed herein. Another general class of zupar1 analogs
is provided by anti-idiotype antibodies, and fragments thereof.
Moreover, recombinant antibodies comprising anti-idiotype variable
domains can be used as analogs (see, for example, Monfardini et
al., Proc. Assoc. Am. Physicians 108:420 (1996)). Since the
variable domains of anti-idiotype zupar1 antibodies mimic zupar1,
these domains can provide either zupar1 agonist or antagonist
activity. As an illustration, Lim and Langer, J. Interferon Res.
13:295 (1993), describe anti-idiotypic interferon-.alpha.
antibodies that have the properties of either interferon-.alpha.
agonists or antagonists.
[0213] Another approach to identifying zupar1 analogs is provided
by the use of combinatorial libraries. Methods for constructing and
screening phage display and other combinatorial libraries are
provided, for example, by Kay et al., Phage Display of Peptides and
Proteins (Academic Press 1996), Verdine, U.S. Pat. No. 5,783,384,
Kay, et. al., U.S. Pat. No. 5,747,334, and Kauffman et al., U.S.
Pat. No. 5,723,323.
[0214] Solution in vitro assays can be used to identify a zupar1
ligand. Solid phase systems can also be used to identify a target
of a zupar1 polypeptide. For example, a zupar1 polypeptide or
zupar1 fusion protein can be immobilized onto the surface of a
receptor chip of a biosensor instrument (BIACORE, Biacore AB;
Uppsala, Sweden). The use of this instrument is disclosed, for
example, by Karlsson, Immunol. Methods 145:229 (1991). In brief, a
zupar1 polypeptide or fusion protein is covalently attached, using
amine or sulfhydryl chemistry, to dextran fibers that are attached
to gold film within a flow cell. A test sample is then passed
through the cell. If a zupar1 target molecule is present in the
sample, it will bind to the immobilized polypeptide or fusion
protein, causing a change in the refractive index of the medium,
which is detected as a change in surface plasmon resonance of the
gold film. This system allows the determination on- and off-rates,
from which binding affinity can be calculated, and assessment of
the stoichiometry of binding, as well as the kinetic effects of
zupar1 mutation.
[0215] In another approach, proteins and peptides of the present
invention can be immobilized on a column and used to bind ligands
from biological preparations that are run over the column
(Hermanson et al. (eds.), Immobilized Affinity Ligand Techniques,
pages 195-202 (Academic Press 1992)). Radiolabeled or affinity
labeled zupar1 polypeptides can also be used to identify or to
localize zupar1 targets in a biological sample (see, for example,
Deutscher (ed.), Methods in Enzymol., vol. 182, pages 721-37
(Academic Press 1990); Brunner et al., Ann. Rev. Biochem. 62:483
(1993); Fedan et al., Biochem. Pharmacol. 33:1167 (1984)). Also
see, Varthakavi and Minocha, J. Gen. Virol. 77:1875 (1996), who
describe the use of anti-idiotype antibodies for receptor
identification.
[0216] Soluble fragments of zupar1 polypeptides, variants, and
zupar1 antibodies could be used to compete with complement to
inhibit inflammation. Complement and C1q play a role in
inflammation. Complement activation is initiated either by the
classical pathway which requires an antigen-antibody interaction
and C1q binding (complement fixation) or an alternative pathway
that occurs when plasmid proteins interact with microorganism cell
wall polysaccharides. Complement proteins are then activated in a
cascade that results in the cleavage of C3 into C3a and C3b. C3b
then binds the target cell surface and triggers insertion of a
membrane attack complex that forms a pore in the membrane,
resulting in lysis of the of the cell. Inhibitors of C1q and
complement would be useful as anti-inflammatory agents. The
complement binding activity of zupar1 can be measured using methods
known in the art, for example, a .sup.51Cr-release assay (Davies et
al., J. Exp. Med. 170:637-54, 1989) or a Calcein release assay
(Bodian et al., J. Exp. Med. 185:507-16, 1997). The complement
binding activity of soluble zupar1 can be compared with soluble
forms of a related protein, CD59, and antibodies to CD59, known to
regulate complement activation by binding to the C5b-8 or C5b-9 of
the membrane attack complex (Davies et al., ibid, and Bodian et
al., ibid).
[0217] In a related application, zupar1 polypeptides, fragments,
fusion proteins, antibodies, or agonists can used in wound healing.
Wound-healing models include the linear skin incision model of
Mustoe et al. (Science 237:1333, 1987). In a typical procedure, a
6-cm incision is made in the dorsal pelt of an adult rat, then
closed with wound clips. Test substances and controls (in solution,
gel, or powder form) are applied before primary closure. It is
preferred to limit administration to a single application, although
additional applications can be made on succeeding days by careful
injection at several sites under the incision. Wound breaking
strength is evaluated between 3 and 21 days post wounding. In a
second model, multiple, small, full-thickness excisions are made on
the ear of a rabbit. The cartilage in the ear splints the wound,
removing the variable of wound contraction from the evaluation of
closure. Experimental treatments and controls are applied. The
geometry and anatomy of the wound site allow for reliable
quantification of cell ingrowth and epithelial migration, as well
as quantitative analysis of the biochemistry of the wounds (e.g.,
collagen content). See, Mustoe et al., J. Clin. Invest. 87:694,
1991. The rabbit ear model can be modified to create an ischemic
wound environment, which more closely resembles the clinical
situation (Ahn et al., Ann. Plast. Surg. 24:17, 1990). Within a
third model, healing of partial-thickness skin wounds in pigs or
guinea pigs is evaluated (LeGrand et al., Growth Factors 8:307,
1993). Experimental treatments are applied daily on or under
dressings. Seven days after wounding, granulation tissue thickness
is determined. This model is preferred for dose-response studies,
as it is more quantitative than other in vivo models of wound
healing. A full thickness excision model can also be employed.
Within this model, the epidermis and dermis are removed down to the
panniculus carnosum in rodents or the subcutaneous fat in pigs.
Experimental treatments are applied topically on or under a
dressing, and can be applied daily if desired. The wound closes by
a combination of contraction and cell ingrowth and proliferation.
Measurable endpoints include time to wound closure, histologic
score, and biochemical parameters of wound tissue. Impaired wound
healing models are also known in the art (e.g., Cromack et al.,
Surgery 113:36, 1993; Pierce et al., Proc. Natl. Acad. Sci. USA
86:2229, 1989; Greenhalgh et al., Amer. J. Pathol 136:1235, 1990).
Delay or prolongation of the wound healing process can be induced
pharmacologically by treatment with steroids, irradiation of the
wound site, or by concomitant disease states (e.g., diabetes).
Linear incisions or full-thickness excisions are most commonly used
as the experimental wound. Endpoints are as disclosed above for
each type of wound. Subcutaneous implants can be used to assess
compounds acting in the early stages of wound healing (Broadley et
al., Lab. Invest. 61:571, 1985; Sprugel et al., Amer. J. Pathol.
129: 601, 1987). Implants are prepared in a porous, relatively
non-inflammatory container (e.g., polyethylene sponges or expanded
polytetrafluoroethylene implants filled with bovine collagen) and
placed subcutaneously in mice or rats. The interior of the implant
is empty of cells, producing a "wound space" that is well-defined
and separable from the preexisting tissue. This arrangement allows
the assessment of cell influx and cell type as well as the
measurement of vasculogenesis/angiogenesis and extracellular matrix
production.
[0218] Zupar1 exhibits homology to known plasminogen activator
proteins. Pro-fibrinolytic modulation can be measured for soluble
zupar1 polypeptide fragments, variants, agonists. Blood coagulation
is a process consisting of a complex interaction of various blood
components which eventually give rise to a fibrin clot. Degradation
of the fibrin clot can be accomplished by activation of plasminogen
into plasmin. Such activators include urokinase plasminogen
activators (uPA). Solublized receptors for these proteins and
antibodies to such receptors are useful for breaking down clots.
Increased plasminogen activator activity is also associated with
metastatic tumors, Min et al., J. Immuno. 148:3636-42, 1992. Zupar1
modulation of fibrinolysis can be measured in a rat model wherein
the enzyme batroxobin and radiolabeled fibrinogen are administered
to test animals. Inhibition of fibrinogen activation by a test
compound is seen as a reduction in the circulating level of the
label as compared to animals not receiving the test compound. See,
Lenfors and Gustafsson, Semin. Thromb. Hemost. 22:335-342,
1996.
[0219] Zupar1 is highly expressed in testis tissue indicating an
association with spermatogenesis. Spermatogenesis is a sequential
process and takes place in the seminiferous tubules, where germ
cells ultimately mature into spermatozoa, herein referred to as
sperm. Testis-specific factors that influence the maturation
process may come directly from the Sertoli cells that are in
contact with spermatogenic cells, or form the Leydig cells that are
located in the interstitial space between the seminiferous tubules.
The sperm is then transported to the epididymus, where sperm
motility and fertilization capacity are increased. The sperm is
stored in the tail of the epididymus until released.
[0220] Before reaching the oocyte or egg and initiating an
egg-sperm interaction, the sperm must be activated. Sperm undergo a
gradual capacitation, lasting up to 3 or 4 hours in vitro, during
which the plasma membrane of the sperm head and the outer acrosomal
membrane fuse to form vesicles that facilitate the release of
acrosomal enzymes. The acrosomal membrane surrounds the acrosome or
acrosomal cap which is located at the anterior end of the nucleus
in the sperm head.
[0221] In order for the sperm to interact with the egg the sperm
must penetrate the oocyte. To enable this process the sperm must
undergo acrosomal exocytosis and release the acrosomal enzymes in
the vicinity of the oocyte. These enzymes enable the sperm to
penetrate the various oocyte layers, (the cumulus oophorus, the
corona radiata and the zona pellucida) and initiate an interaction
with the egg. During the acrosomal reaction, proacrosin is
converted to acrosin, the active form of the enzyme, which is
required for and must occur before binding and penetration of the
zona pellucida is possible (Zaneveld, in Male Infertility Chapter
11, Comhaire (Ed.), Chapman & Hall, London 1996).
[0222] The testis are an immune privileged area and sperm, like
oocytes, are major histocompatibility factor Class I (MHC Class I)
negative. Therefore the sperm, egg, and more importantly the
fertilized egg, need some mechanism to protect them from the innate
immune response of the uterus. One proposed mechanism is the use of
bisecting type glycans on the cell surface of the sperm and egg
which inhibit NK cell function. Zupar1 expression in the testis
likely indicates another immune response inhibitor mechanism.
Zupar1 polypeptides, fragments, agonists, and antibodies would be
of use in enhancing viability of cryopreserved sperm, as a
component in the acrosome reaction, enhancing sperm motility and
enhancing egg-sperm interactions.
[0223] Accordingly, proteins, including soluble proteins and
antibodies, of the present invention may have applications in
enhancing fertilization, particularly during assisted reproduction
in humans and in animals, by preventing attack by complement. Such
assisted reproduction methods are known in the art and include
artificial insemination, in vitro fertilization, embryo transfer
and gamete intrafallopian transfer. Proteins of the present
invention can be combined with sperm, an egg or an egg-sperm
mixture prior to fertilization of the egg. The proteins of the
present invention could be added, for example, to the capacitation
media that is used to activate the sperm. Polypeptides of the
present invention can used in such methods to enhance viability of
cryopreserved sperm.
[0224] In cases where pregnancy is not desired, zupar1 proteins or
protein fragments, and agonists may function as germ-cell-specific
antigens for use as components in "immunocontraceptive" or
"anti-fertility" vaccines to induce formation of antibodies to
selectively inhibit a cell-mediated immunity, a processes critical
to successful reproduction in humans and animals. The use of sperm
and testis antigens in the development of an immunocontraceptive
have been described (O'Hern et al., Biol Reprod. 52:311-39, 1995;
Diekman and Herr, Am. J. Reprod. Immunol. 37:111-17, 1997; Zhu and
Naz, Proc. Natl. Acad. Sci. USA 94:4704-9,1997).
[0225] Zupar1 antibodies could also act as antagonists by
inhibiting components of spermatogenesis and/or sperm activation.
Such antibody "antagonists" can be used for contraception in
animals, and in particular, domestic animals and livestock. For
instance, anti-zupar1 immunization could be used in place of
surgical forms of contraception (such as spaying and neutering),
and would allow for the possibility of future breeding if
desired.
[0226] The effect of zupar1 on testes can be determined by methods
known in the art and described herein. Zupar1 can be injected
intraperitoneally for a specific time duration. After the treatment
period, animals are sacrificed and testes removed and weighed.
Testicles are homogenized and sperm head counts are made (Meistrich
et al., Exp. Cell Res. 99:72-78, 1976).
[0227] Zupar1 polypeptides, fragments, and antibodies would be
useful as markers for testis tissue or sperm. Use of such markers
is known in the art and tissue markers are available
commercially.
[0228] Zupar1 polypeptides, fragments, and agonist would useful as
components of defined cell culture media, and may be used alone or
in combination with other cytokines and hormones to replace serum
that is commonly used in cell culture for stimulating proliferation
and development of target cells in vitro and in vivo. Cultured
testicular cells include dolphin DB1.Tes cells (CRL-6258); mouse
GC-1 spg cells (CRL-2053); TM3 cells (CRL-1714); TM4 cells
(CRL-1715); and pig ST cells (CRL-1746), available from American
Type Culture Collection, Manassas, Va. Proliferation and
differentiation can be measured using cultured testicular cells or
in vivo by administering molecules of the present invention to the
appropriate animal model. Assays measuring cell proliferation or
differentiation are well known in the art. For example, assays
measuring proliferation include such assays as chemosensitivity to
neutral red dye (Cavanaugh et al., Investigational New Drugs
8:347-354, 1990), incorporation of radiolabelled nucleotides (Cook
et al., Analytical Biochem. 179:1-7, 1989), incorporation of
5-bromo-2'-deoxyuridine (BrdU) in the DNA of proliferating cells
(Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use
of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55-63, 1983;
Alley et al., Cancer Res. 48:589-601, 1988; Marshall et al., Growth
Reg. 5:69-84, 1995; and Scudiero et al., Cancer Res. 48:4827-4833,
1988). Assays measuring differentiation include, for example,
measuring cell-surface markers associated with stage-specific
expression of a tissue, enzymatic activity, functional activity or
morphological changes (Watt, FASEB, 5:281-284, 1991; Francis,
Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol.
Technol. Bioprocesses, 161-171, 1989).
[0229] In addition to the uses described above, polynucleotides and
polypeptides of the present invention are useful as educational
tools in laboratory practicum kits for courses related to genetics
and molecular biology, protein chemistry, and antibody production
and analysis. Due to its unique polynucleotide and polypeptide
sequences, molecules of zupar1 can be used as standards or as
"unknowns" for testing purposes. For example, zupar1
polynucleotides can be used as an aid, such as, for example, to
teach a student how to prepare expression constructs for bacterial,
viral, or mammalian expression, including fusion constructs,
wherein zupar1 is the gene to be expressed; for determining the
restriction endonuclease cleavage sites of the polynucleotides;
determining mRNA and DNA localization of zupar1 polynucleotides in
tissues (i.e., by northern and Southern blotting as well as
polymerase chain reaction); and for identifying related
polynucleotides and polypeptides by nucleic acid hybridization.
[0230] Zupar1 polypeptides can be used as an aid to teach
preparation of antibodies; identifying proteins by western
blotting; protein purification; determining the weight of expressed
zupar1 polypeptides as a ratio to total protein expressed;
identifying peptide cleavage sites; coupling amino and carboxyl
terminal tags; amino acid sequence analysis, as well as, but not
limited to monitoring biological activities of both the native and
tagged protein (i.e., protease inhibition) in vitro and in
vivo.
[0231] Zupar1 polypeptides can also be used to teach analytical
skills such as mass spectrometry, circular dichroism to determine
conformation, especially of the single alpha helix and the five
beta strands, x-ray crystallography to determine the
three-dimensional structure in atomic detail, nuclear magnetic
resonance spectroscopy to reveal the structure of proteins in
solution. Identification of the 10 cysteine residues and 5
disulfide bonds can be determined, for example, as well as the
active site residue. For example, a kit containing the zupar1 can
be given to the student to analyze. Since the amino acid sequence
would be known by the instructor, the protein can be given to the
student as a test to determine the skills or develop the skills of
the student, the instructor would then know whether or not the
student has correctly analyzed the polypeptide. Since every
polypeptide is unique, the educational utility of zupar1 would be
unique unto itself.
[0232] The antibodies which bind specifically to zupar1 can be used
as a teaching aid to instruct students how to prepare affinity
chromatography columns to purify zupar1, cloning and sequencing the
polynucleotide that encodes an antibody and thus as a practicum for
teaching a student how to design humanized antibodies. The zupar1
gene, polypeptide, or antibody would then be packaged by reagent
companies and sold to educational institutions so that the students
gain skill in art of molecular biology. Because each gene and
protein is unique, each gene and protein creates unique challenges
and learning experiences for students in a lab practicum. Such
educational kits containing the zupar1 gene, polypeptide, or
antibody are considered within the scope of the present
invention.
[0233] 10. Use of zupar1 Nucleotide Sequences to Detect zupar1 Gene
Expression and to Examine zupar1 Gene Structure
[0234] Nucleic acid molecules can be used to detect the expression
of a zupar1 gene in a biological sample. Such probe molecules
include double-stranded nucleic acid molecules comprising the
nucleotide sequence of SEQ ID NO:1, or a portion thereof, as well
as single-stranded nucleic acid molecules having the complement of
the nucleotide sequence of SEQ ID NO:1, or a portion thereof. Probe
molecules may be DNA, RNA, oligonucleotides, and the like. As used
herein, the term "portion" refers to at least eight nucleotides to
at least 20 or more nucleotides. Certain probes bind with regions
of the zupar1 gene that have a low sequence similarity to
comparable regions in other urokinase plasminogen activator,
vasopressin-neurophysin, or CD59 genes.
[0235] In a basic assay, a single-stranded probe molecule is
incubated with RNA, isolated from a biological sample, under
conditions of temperature and ionic strength that promote base
pairing between the probe and target zparl RNA species. After
separating unbound probe from hybridized molecules, the amount of
hybrids is detected.
[0236] Well-established hybridization methods of RNA detection
include northern analysis and dot/slot blot hybridization (see, for
example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.),
"Analysis of Gene Expression at the RNA Level," in Methods in Gene
Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid
probes can be detectably labeled with radioisotopes such as
.sup.32P or .sup.35S. Alternatively, zupar1 RNA can be detected
with a nonradioactive hybridization method (see, for example, Isaac
(ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes
(Humana Press, Inc. 1993)). Typically, nonradioactive detection is
achieved by enzymatic conversion of chromogenic or chemiluminescent
substrates. Illustrative nonradioactive moieties include biotin,
fluorescein, and digoxigenin.
[0237] Zupar1 oligonucleotide probes are also useful for in vivo
diagnosis. As an illustration, .sup.18F-labeled oligonucleotides
can be administered to a subject and visualized by positron
emission tomography (Tavitian et al., Nature Medicine 4:467
(1998)).
[0238] Numerous diagnostic procedures take advantage of the
polymerase chain reaction (PCR) to increase sensitivity of
detection methods. Standard techniques for performing PCR are
well-known (see, generally, Mathew (ed.), Protocols in Human
Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR
Protocols: Current Methods and Applications (Humana Press, Inc.
1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press,
Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols
(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR
(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis
(Humana Press, Inc. 1998)). Particular PCR primers are designed to
amplify a portion of the zupar1 gene that has a low sequence
similarity to a comparable region in other genes encoding membrane
bound proteins.
[0239] One variation of PCR for diagnostic assays is reverse
transcriptase-PCR (RT-PCR). hi the RT-PCR technique, RNA is
isolated from a biological sample, reverse transcribed to cDNA, and
the cDNA is incubated with zupar1 primers (see, for example, Wu et
al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in
Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)).
PCR is then performed and the products are analyzed using standard
techniques.
[0240] As an illustration, RNA is isolated from biological sample
using, for example, the guanidinium-thiocyanate cell lysis
procedure described above. Alternatively, a solid-phase technique
can be used to isolate mRNA from a cell lysate. A reverse
transcription reaction can be primed with the isolated RNA using
random oligonucleotides, short homopolymers of dT, or zupar1
anti-sense oligomers. Oligo-dT primers offer the advantage that
various mRNA nucleotide sequences are amplified that can provide
control target sequences. zupar1 sequences are amplified by the
polymerase chain reaction using two flanking oligonucleotide
primers that are typically 20 bases in length.
[0241] PCR amplification products can be detected using a variety
of approaches. For example, PCR products can be fractionated by gel
electrophoresis, and visualized by ethidium bromide staining.
Alternatively, fractionated PCR products can be transferred to a
membrane, hybridized with a detectably-labeled zupar1 probe, and
examined by autoradiography. Additional alternative approaches
include the use of digoxigenin-labeled deoxyribonucleic acid
triphosphates to provide chemiluminescence detection, and the
C-TRAK colorimetric assay.
[0242] Another approach for detection of zupar1 expression is
cycling probe technology, in which a single-stranded DNA target
binds with an excess of DNA-RNA-DNA chimeric probe to form a
complex, the RNA portion is cleaved with RNAase H, and the presence
of cleaved chimeric probe is detected (see, for example, Beggs et
al., J. Clin. Microbiol. 34:2985 (1996), Bekkaoui et al.,
Biotechniques 20:240 (1996)). Alternative methods for detection of
zupar1 sequences can utilize approaches such as nucleic acid
sequence-based amplification, cooperative amplification of
templates by cross-hybridization, and the ligase chain reaction
(see, for example, Marshall et al., U.S. Pat. No. 5,686,272 (1997),
Dyer et al., J. Virol. Methods 60:161 (1996), Ehricht et al., Eur.
J. Biochem. 243:358 (1997), and Chadwick et al., J. Virol. Methods
70:59 (1998)). Other standard methods are known to those of skill
in the art.
[0243] Zupar1 probes and primers can be used to detect and to
localize zupar1 gene expression in tissue samples. Methods for such
in situ hybridization are well-known to those of skill in the art
(see, for example, Choo (ed.), In Situ Hybridization Protocols
(Humana Press, Inc. 1994), Wu et al. (eds.), "Analysis of Cellular
DNA or Abundance of mRNA by Radioactive In Situ Hybridization
(RISH)," in Methods in Gene Biotechnology, pages 259-278 (CRC
Press, Inc. 1997), and Wu et al. (eds.), "Localization of DNA or
Abundance of mRNA by Fluorescence In Situ Hybridization (RISH)," in
Methods in Gene Biotechnology, pages 279-289 (CRC Press, Inc.
1997)). Various additional diagnostic approaches are well-known to
those of skill in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc.
1996), and Elles, Molecular Diagnosis of Genetic Diseases (Humana
Press, Inc., 1996)).
[0244] Aberrations associated with the zupar1 locus can be detected
using nucleic acid molecules of the present invention by employing
molecular genetic techniques, such as restriction fragment length
polymorphism analysis, short tandem repeat analysis employing PCR
techniques, amplification-refractory mutation system analysis,
single-strand conformation polymorphism detection, RNase cleavage
methods, denaturing gradient gel electrophoresis,
fluorescence-assisted mismatch analysis, and other genetic analysis
techniques known in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Marian, Chest 108:255 (1995), Coleman and Tsongalis, Molecular
Diagnostics (Human Press, Inc. 1996), Elles (ed.) Molecular
Diagnosis of Genetic Diseases (Humana Press, Inc. 1996), Landegren
(ed.), Laboratory Protocols for Mutation Detection (Oxford
University Press 1996), Birren et al. (eds.), Genome Analysis, Vol.
2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),
Dracopoli et al. (eds.), Current Protocols in Human Genetics (John
Wiley & Sons 1998), and Richards and Ward, "Molecular
Diagnostic Testing," in Principles of Molecular Medicine, pages
83-88 (Humana Press, Inc. 1998)).
[0245] The protein truncation test is also useful for detecting the
inactivation of a gene in which translation-terminating mutations
produce only portions of the encoded protein (see, for example,
Stoppa-Lyonnet et al., Blood 91:3920 (1998)). According to this
approach, RNA is isolated from a biological sample, and used to
synthesize cDNA. PCR is then used to amplify the zupar1 target
sequence and to introduce an RNA polymerase promoter, a translation
initiation sequence, and an inframe ATG triplet. PCR products are
transcribed using an RNA polymerase, and the transcripts are
translated in vitro with a T7-coupled reticulocyte lysate system.
The translation products are then fractionated by SDS-PAGE to
determine the lengths of the translation products. The protein
truncation test is described, for example, by Dracopoli et al.
(eds.), Current Protocols in Human Genetics, pages 9.11.1-9.11.18
(John Wiley & Sons 1998).
[0246] The present invention also contemplates kits for performing
an assay to detect zupar1 gene expression, or to examine the zupar1
locus. Such kits comprise nucleic acid probes, such as
double-stranded nucleic acid molecules comprising the nucleotide
sequence of SEQ ID NO:1, or a portion thereof, as well as
single-stranded nucleic acid molecules having the complement of the
nucleotide sequence of SEQ ID NO:1, or a portion thereof. As an
illustration, a suitable probe comprises the nucleotide sequence of
SEQ ID NO:1. Probe molecules may be DNA, RNA, oligonucleotides, and
the like. Kits may comprise nucleic acid primers for performing
PCR.
[0247] Such a kit can contain all the necessary elements to perform
a nucleic acid assay described above. A kit will comprise at least
one container comprising a zupar1 probe or primer. The kit may also
comprise a second container comprising one or more reagents capable
of indicating the presence of zupar1 sequences. Examples of such
indicator reagents include detectable labels such as radioactive
labels, fluorochromes, chemiluminescent agents, and the like. A kit
may also comprise a means for conveying to the user that the zupar1
probes and primers are used to detect zupar1 gene expression. For
example, written instructions may state that the enclosed nucleic
acid molecules can be used to detect either a nucleic acid molecule
that encodes zupar1, or a nucleic acid molecule having a nucleotide
sequence that is complementary to a zupar1-encoding nucleotide
sequence. The written material can be applied directly to a
container, or the written material can be provided in the form of a
packaging insert.
[0248] 11. Use of Anti-zupar1 Antibodies to Detect zupar1
Protein
[0249] The present invention contemplates the use of anti-zupar1
antibodies to screen biological samples in vitro for the presence
of zupar1. In one type of in vitro assay, anti-zupar1 antibodies
are used in liquid phase. For example, the presence of zupar1 in a
biological sample can be tested by mixing the biological sample
with a trace amount of labeled zupar1 and an anti-zupar1 antibody
under conditions that promote binding between zupar1 and its
antibody. Complexes of zupar1 and anti-zupar1 in the sample can be
separated from the reaction mixture by contacting the complex with
an immobilized protein which binds with the antibody, such as an Fc
antibody or Staphylococcus protein A. The concentration of zupar1
in the biological sample will be inversely proportional to the
amount of labeled zupar1 bound to the antibody and directly related
to the amount of free labeled zupar1.
[0250] Alternatively, in vitro assays can be performed in which
anti-zupar1 antibody is bound to a solid-phase carrier. For
example, antibody can be attached to a polymer, such as
aminodextran, in order to link the antibody to an insoluble support
such as a polymer-coated bead, a plate or a tube. Other suitable in
vitro assays will be readily apparent to those of skill in the
art.
[0251] In another approach, anti-zupar1 antibodies can be used to
detect zupar1 in tissue sections prepared from a biopsy specimen.
Such immunochemical detection can be used to determine the relative
abundance of zupar1 and to determine the distribution of zupar1 in
the examined tissue. General immunochemistry techniques are well
established (see, for example, Ponder, "Cell Marking Techniques and
Their Application," in Mammalian Development: A Practical Approach,
Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages
5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley
Interscience 1990), and Manson (ed.), Methods In Molecular Biology,
Vol. 10: Immunochemical Protocols (The Humana Press, Inc.
1992)).
[0252] Immunochemical detection can be performed by contacting a
biological sample with an anti-zupar1 antibody, and then contacting
the biological sample with a detectably labeled molecule which
binds to the antibody. For example, the detectably labeled molecule
can comprise an antibody moiety that binds to anti-zupar1 antibody.
Alternatively, the anti-zupar1 antibody can be conjugated with
avidin/streptavidin (or biotin) and the detectably labeled molecule
can comprise biotin (or avidin/streptavidin). Numerous variations
of this basic technique are well-known to those of skill in the
art.
[0253] Alternatively, an anti-zupar1 antibody can be conjugated
with a detectable label to form an anti-zupar1 immunoconjugate.
Suitable detectable labels include, for example, a radioisotope, a
fluorescent label, a chemiluminescent label, an enzyme label, a
bioluminescent label or colloidal gold. Methods of making and
detecting such detectably-labeled immunoconjugates are well-known
to those of ordinary skill in the art, and are described in more
detail below.
[0254] The detectable label can be a radioisotope that is detected
by autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0255] Anti-zupar1 immunoconjugates can also be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody is determined by exposing the immunoconjugate to light of
the proper wavelength and detecting the resultant fluorescence.
Fluorescent labeling compounds include fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine.
[0256] Alternatively, anti-zupar1 immunoconjugates can be
detectably labeled by coupling an antibody component to a
chemiluminescent compound. The presence of the
chemiluminescent-tagged immunoconjugate is determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of chemiluminescent labeling compounds
include luminol, isoluminol, an aromatic acridinium ester, an
imidazole, an acridinium salt and an oxalate ester.
[0257] Similarly, a bioluminescent compound can be used to label
anti-zupar1 immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0258] Alternatively, anti-zupar1 immunoconjugates can be
detectably labeled by linking an anti-zupar1 antibody component to
an enzyme. When the anti-zupar1enzyme conjugate is incubated in the
presence of the appropriate substrate, the enzyme moiety reacts
with the substrate to produce a chemical moiety which can be
detected, for example, by spectrophotometric, fluorometric or
visual means. Examples of enzymes that can be used to detectably
label polyspecific immunoconjugates include .beta.-galactosidase,
glucose oxidase, peroxidase and alkaline phosphatase.
[0259] Those of skill in the art will know of other suitable
labels, which can be employed in accordance with the present
invention. The binding of marker moieties to anti-zupar1 antibodies
can be accomplished using standard techniques known to the art.
Typical methodology in this regard is described by Kennedy et al.,
Clin. Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1
(1977), Shih et al., Int'l J. Cancer 46:1101 (1990), Stein etal.,
Cancer Res. 50:1330 (1990), and Coligan, supra.
[0260] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-zupar1 antibodies that have
been conjugated with avidin, streptavidin, and biotin (see, for
example, Wilchek et al. (eds.), "Avidin-Biotin Technology," Methods
In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,
"Immunochemical Applications of Avidin-Biotin Technology," in
Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162
(The Humana Press, Inc. 1992).
[0261] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic immunoassays," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0262] In a related approach, biotin- or FITC-labeled zupar1 can be
used to identify cells that bind zupar1. Such can binding can be
detected, for example, using flow cytometry.
[0263] The present invention contemplates kits for detecting the
presence of zupar1. Such kits comprise at least one container
comprising an anti-zupar1 antibody, or antibody fragment. A kit may
also comprise a second container comprising one or more reagents
capable of indicating the presence of zupar1 antibody or antibody
fragments. Examples of such indicator reagents include detectable
labels such as a radioactive label, a fluorescent label, a
chemiluminescent label, an enzyme label, a bioluminescent label,
colloidal gold, and the like. A kit may also comprise a means for
conveying to the user that zupar1 antibodies or antibody fragments
are used to detect zupar1 protein. For example, written
instructions may state that the enclosed antibody or antibody
fragment can be used to detect zupar1. The written material can be
applied directly to a container, or the written material can be
provided in the form of a packaging insert.
[0264] 12. Production of Transgenic Mice
[0265] Transgenic mice can be engineered to over-express the zupar1
gene in all tissues or under the control of a tissue-specific or
tissue-preferred regulatory element. These over-producers of zupar1
can be used to characterize the phenotype that results from
over-expression, and the transgenic animals can serve as models for
human disorders. Transgenic mice that over-express zupar1 also
provide model bioreactors for production of zupar1 in the milk or
blood of larger animals. Methods for producing transgenic mice are
well-known to those of skill in the art (see, for example, Jacob,
"Expression and Knockout of Interferons in Transgenic Mice," in
Overexpression and Knockout of Cytokines in Transgenic Mice, Jacob
(ed.), pages 111-124 (Academic Press, Ltd. 1994), Monastersky and
Robl (eds.), Strategies in Transgenic Animal Science (ASM Press
1995), and Abbud and Nilson, "Recombinant Protein Expression in
Transgenic Mice," in Gene Expression Systems: Using Nature for the
Art of Expression, Fernandez and Hoeffler (eds.), pages 367-397
(Academic Press, Inc. 1999)).
[0266] For example, a method for producing a transgenic mouse that
expresses a zupar1 gene can begin with adult, fertile males (studs)
(B6C3f1, 2-8 months of age (Taconic Farms, Germantown, N.Y.)),
vasectomized males (duds) (B6D2f1, 2-8 months, (Taconic Farms)),
prepubescent fertile females (donors) (B6C3f1, 4-5 weeks, (Taconic
Farms)) and adult fertile females (recipients) (B6D2f1, 2-4 months,
(Taconic Farms)). The donors are acclimated for one week and then
injected with approximately 8 IU/mouse of Pregnant Mare's Serum
gonadotrophin (Sigma Chemical Company; St. Louis, Mo.) I.P., and
46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG
(Sigma)) I.P. to induce superovulation. Donors are mated with studs
subsequent to hormone injections. Ovulation generally occurs within
13 hours of hCG injection. Copulation is confirmed by the presence
of a vaginal plug the morning following mating. Fertilized eggs are
collected under a surgical scope. The oviducts are collected and
eggs are released into urinanalysis slides containing hyaluronidase
(Sigma). Eggs are washed once in hyaluronidase, and twice in
Whitten's W640 medium (described, for example, by Menino and
O'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs,
Zygote 4:129 (1996)) that has been incubated with 5% CO.sub.2, 5%
O.sub.2, and 90% N.sub.2 at 37.degree. C. The eggs are then stored
in a 37.degree. C./5% CO.sub.2 incubator until microinjection.
[0267] Ten to twenty micrograms of plasmid DNA containing a zupar1
encoding sequence is linearized, gel-purified, and resuspended in
10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final
concentration of 5-10 nanograms per microliter for microinjection.
For example, the zupar1 encoding sequences can encode a polypeptide
comprising the amino acid residue sequence of SEQ ID NO:2.
[0268] Plasmid DNA is microinjected into harvested eggs contained
in a drop of W640 medium overlaid by warm, CO.sub.2-equilibrated
mineral oil. The DNA is drawn into an injection needle (pulled from
a 0.75 mm ID, 1 mm OD borosilicate glass capillary), and injected
into individual eggs. Each egg is penetrated with the injection
needle, into one or both of the haploid pronuclei.
[0269] Picoliters of DNA are injected into the pronuclei, and the
injection needle withdrawn without coming into contact with the
nucleoli. The procedure is repeated until all the eggs are
injected. Successfully microinjected eggs are transferred into an
organ tissue-culture dish with pre-gassed W640 medium for storage
overnight in a 37.degree. C./5% CO.sub.2 incubator.
[0270] The following day, two-cell embryos are transferred into
pseudopregnant recipients. The recipients are identified by the
presence of copulation plugs, after copulating with vasectomized
duds. Recipients are anesthetized and shaved on the dorsal left
side and transferred to a surgical microscope. A small incision is
made in the skin and through the muscle wall in the middle of the
abdominal area outlined by the ribcage, the saddle, and the hind
leg, midway between knee and spleen. The reproductive organs are
exteriorized onto a small surgical drape. The fat pad is stretched
out over the surgical drape, and a baby serrefine (Roboz,
Rockville, Md.) is attached to the fat pad and left hanging over
the back of the mouse, preventing the organs from sliding back
in.
[0271] With a fine transfer pipette containing mineral oil followed
by alternating W640 and air bubbles, 12-17 healthy two-cell embryos
from the previous day's injection are transferred into the
recipient. The swollen ampulla is located and holding the oviduct
between the ampulla and the bursa, a nick in the oviduct is made
with a 28 g needle close to the bursa, making sure not to tear the
ampulla or the bursa.
[0272] The pipette is transferred into the nick in the oviduct, and
the embryos are blown in, allowing the first air bubble to escape
the pipette. The fat pad is gently pushed into the peritoneum, and
the reproductive organs allowed to slide in. The peritoneal wall is
closed with one suture and the skin closed with a wound clip. The
mice recuperate on a 37.degree. C. slide warmer for a minimum of
four hours.
[0273] The recipients are returned to cages in pairs, and allowed
19-21 days gestation. After birth, 19-21 days postpartum is allowed
before weaning. The weanlings are sexed and placed into separate
sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off
the tail with clean scissors.
[0274] Genomic DNA is prepared from the tail snips using, for
example, a QIAGEN DNEASY kit following the manufacturer's
instructions. Genomic DNA is analyzed by PCR using primers designed
to amplify a zupar1 gene or a selectable marker gene that was
introduced in the same plasmid. After animals are confirmed to be
transgenic, they are back-crossed into an inbred strain by placing
a transgenic female with a wild-type male, or a transgenic male
with one or two wild-type female(s). As pups are born and weaned,
the sexes are separated, and their tails snipped for
genotyping.
[0275] To check for expression of a transgene in a live animal, a
partial hepatectomy is performed. A surgical prep is made of the
upper abdomen directly below the zyphoid process. Using sterile
technique, a small 1.5-2 cm incision is made below the sternum and
the left lateral lobe of the liver exteriorized. Using 4-0 silk, a
tie is made around the lower lobe securing it outside the body
cavity. An atraumatic clamp is used to hold the tie while a second
loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed
proximal to the first tie. A distal cut is made from the Dexon tie
and approximately 100 mg of the excised liver tissue is placed in a
sterile petri dish. The excised liver section is transferred to a
14 ml polypropylene round bottom tube and snap frozen in liquid
nitrogen and then stored on dry ice. The surgical site is closed
with suture and wound clips, and the animal's cage placed on a
37.degree. C. heating pad for 24 hours post operatively. The animal
is checked daily post operatively and the wound clips removed 7-10
days after surgery. The expression level of zupar1 mRNA is examined
for each transgenic mouse using an RNA solution hybridization assay
or polymerase chain reaction.
[0276] In addition to producing transgenic mice that over-express
zupar1, it is useful to engineer transgenic mice with either
abnormally low or no expression of the gene. Such transgenic mice
provide useful models for diseases associated with a lack of a
zupar1 ortholog. As discussed above, zupar1 gene expression can be
inhibited using anti-sense genes, ribozyme genes, or external guide
sequence genes. To produce transgenic mice that under-express the
zupar1 gene, such inhibitory sequences are targeted to zupar1 mRNA.
Methods for producing transgenic mice that have abnormally low
expression of a particular gene are known to those in the art (see,
for example, Wu et al., "Gene Underexpression in Cultured Cells and
Animals by Antisense DNA and RNA Strategies," in Methods in Gene
Biotechnology, pages 205-224 (CRC Press 1997)).
[0277] An alternative approach to producing transgenic mice that
have little or no zupar1 gene expression is to generate mice having
at least one normal zupar1 allele replaced by a nonfunctional
zupar1 gene. One method of designing a nonfunctional zupar1 gene is
to insert another gene, such as a selectable marker gene, within a
nucleic acid molecule that encodes zupar1. Standard methods for
producing these so-called "knockout mice" are known to those
skilled in the art (see, for example, Jacob, "Expression and
Knockout of Interferons in Transgenic Mice," in Overexpression and
Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages
111-124 (Academic Press, Ltd. 1994), and Wu et al., "New Strategies
for Gene Knockout," in Methods in Gene Biotechnology, pages 339-365
(CRC Press 1997)).
[0278] 13. Therapeutic Uses of zupar1 Polypeptides
[0279] The present invention includes the use of proteins,
polypeptides, and peptides having zupar1 activity (such as zupar1
polypeptides, anti-idiotype anti-zupar1 antibodies, and zupar1
fusion proteins) to a subject who lacks an adequate amount of this
polypeptide. In contrast, zupar1 antagonists (e.g., anti- zupar1
antibodies) can be used to treat a subject who produces an excess
of zupar1. These molecules can be administered to any subject in
need of treatment, and the present invention contemplates both
veterinary and human therapeutic uses. Illustrative subjects
include mammalian subjects, such as farm animals, zoological
animals, show and sports animals, domestic animals, and human
patients.
[0280] Proteins, polypeptides, and peptides having zupar1 activity
could be exploited in therapeutic regimes, for applications related
to inflammation, fibrinolysis, coagulation, remodeling, wound
healing contraception, and fertility.
[0281] The dosage of administered polypeptide, protein or peptide
will vary depending upon such factors as the subject's age, weight,
height, sex, general medical condition and previous medical
history. Typically, it is desirable to provide the recipient with a
dosage of a molecule having zupar1 activity, which is in the range
of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of
subject), although a lower or higher dosage also may be
administered as circumstances dictate.
[0282] Administration of a molecule having zupar1 activity to a
subject can be intravenous, intraarterial, intraperitoneal,
intramuscular, subcutaneous, intrapleural, intrathecal, by
perfusion through a regional catheter, or by direct intralesional
injection. When administering therapeutic proteins by injection,
the administration may be by continuous infusion or by single or
multiple boluses.
[0283] A pharmaceutical composition comprising a protein,
polypeptide, or peptide having zupar1 activity can be formulated
according to known methods to prepare pharmaceutically useful
compositions, whereby the therapeutic proteins are combined in a
mixture with a pharmaceutically acceptable carrier. A composition
is said to be a "pharmaceutically acceptable carrier" if its
administration can be tolerated by a recipient subject. Sterile
phosphate-buffered saline is one example of a pharmaceutically
acceptable carrier. Other suitable carriers are well-known to those
in the art. See, for example, Gennaro (ed.), Remington's
Pharmaceutical Sciences, 19th Edition (Mack Publishing Company
1995).
[0284] For purposes of therapy, molecules having zupar1 activity
and a pharmaceutically acceptable carrier are administered to a
subject in a therapeutically effective amount. A combination of a
protein, polypeptide, or peptide having zupar1 activity and a
pharmaceutically acceptable carrier is said to be administered in a
"therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient subject.
[0285] A pharmaceutical composition comprising molecules having
zupar1 activity can be furnished in liquid form, or in solid form.
Liquid forms, including liposome-encapsulated formulations, are
illustrated by injectable solutions and oral suspensions. Exemplary
solid forms include capsules, tablets, and controlled-release
forms, such as a miniosmotic pump or an implant. Other dosage forms
can be devised by those skilled in the art, as shown, for example,
by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug
Delivery Systems, 5.sup.th Edition (Lea & Febiger 1990),
Gennaro (ed.), Remington's Pharmaceutical Sciences, 19.sup.th
Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0286] The present invention also contemplates the use of zupar1
polypeptide-toxin fusion proteins or antibody-toxin fusion proteins
for targeted cell or tissue inhibition or ablation (for instance,
to treat cancer cells or tissues). Such zupar1 polypeptide or
antibody conjugates can be delivered intravenously,
intraarterially, intraductally with dimethylsulfoxide,
intramuscularly, subcutaneously, intraperitoneally, also by
transdermal methods, by electro-transfer, orally or via
inhalant.
[0287] Zupar1 blocking antibodies can also be used to target zupar1
on specific cell populations, inactivating zupar1 and rendering the
cells susceptible to lysis by the complement system.
Complement-dependent lysis can be determined using a
.sup.51Cr-release assay. Cells are labeled with .sup.51Cr and
incubated with zupar1 blocking antibodies, preferably the F(ab')2
fragments. The cells are then exposed to complement. The cells are
centrifuged and the cell pellet lysed. The amount of .sup.51Cr in
the supernatant and cell lysate are then compared. See Brooimans et
al., Eur. J. Immunol. 22:791-7, 1992, and Schmitz et al., J. Clin.
Invest. 95:1520-26, 1995. Zupar1 activity can be compared to other
membrane glycoproteins or GPI-anchored proteins, such as CD59.
[0288] Zupar1 pharmaceutical compositions may be supplied as a kit
comprising a container that comprises zupar1. zupar1 can be
provided in the form of an injectable solution for single or
multiple doses, or as a sterile powder that will be reconstituted
before injection. Such a kit may further comprise written
information on indications and usage of the pharmaceutical
composition.
[0289] 14. Therapeutic Uses of zupar1 Nucleotide Sequences
[0290] The present invention includes the use of zupar1 nucleotide
sequences to provide zupar1 to a subject in need of such treatment.
In addition, a therapeutic expression vector can be provided that
inhibits zupar1 gene expression, such as an anti-sense molecule, a
ribozyme, or an external guide sequence molecule.
[0291] There are numerous approaches to introduce a zupar1 gene to
a subject, including the use of recombinant host cells that express
zupar1, delivery of naked nucleic acid encoding zupar1, use of a
cationic lipid carrier with a nucleic acid molecule that encodes
zupar1, and the use of viruses that express zupar1, such as
recombinant retroviruses, recombinant adeno-associated viruses,
recombinant adenoviruses, and recombinant Herpes simplex viruses
[HSV] (see, for example, Mulligan, Science 260:926 (1993),
Rosenberg et al., Science 242:1575 (1988), LaSalle et al., Science
259:988 (1993), Wolff et al., Science 247:1465 (1990), Breakfield
and Deluca, The New Biologist 3:203 (1991)). In an ex vivo
approach, for example, cells are isolated from a subject,
transfected with a vector that expresses a zupar1 gene, and then
transplanted into the subject.
[0292] In order to effect expression of a zupar1 gene, an
expression vector is constructed in which a nucleotide sequence
encoding a zupar1 gene is operably linked to a core promoter, and
optionally a regulatory element, to control gene transcription. The
general requirements of an expression vector are described
above.
[0293] Alternatively, a zupar1 gene can be delivered using
recombinant viral vectors, including for example, adenoviral
vectors (e.g., Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA
90:11498 (1993), Kolls et al., Proc. Nat'l Acad. Sci. USA 91:215
(1994), Li et al., Hum. Gene Ther. 4:403 (1993), Vincent et al.,
Nat. Genet. 5:130 (1993), and Zabner et al., Cell 75:207 (1993)),
adenovirus-associated viral vectors (Flotte et al., Proc. Nat'l
Acad. Sci. USA 90:10613 (1993)), alphaviruses such as Semliki
Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857
(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,
Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat.
Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus
vectors (Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus
vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993),
Panicali and Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)),
pox viruses, such as canary pox virus or vaccinia virus
(Fisher-Hoch et al., Proc. Nat'l Acad. Sci. USA 86:317 (1989), and
Flexner et al., Ann. N.Y. Acad. Sci. 569:86 (1989)), and
retroviruses (e.g., Baba et al., J. Neurosurg 79:729 (1993), Ram et
al., Cancer Res. 53:83 (1993), Takamiya et al., J. Neurosci. Res
33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993), Vile and
Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S. Pat.
No. 5,399,346). Within various embodiments, either the viral vector
itself, or a viral particle which contains the viral vector may be
utilized in the methods and compositions described below.
[0294] As an illustration of one system, adenovirus, a
double-stranded DNA virus, is a well-characterized gene transfer
vector for delivery of a heterologous nucleic acid molecule (for a
review, see Becker et al., Meth. Cell Biol. 43:161 (1994); Douglas
and Curiel, Science & Medicine 4:44 (1997)). The adenovirus
system offers several advantages including: (i) the ability to
accommodate relatively large DNA inserts, (ii) the ability to be
grown to high-titer, (iii) the ability to infect a broad range of
mammalian cell types, and (iv) the ability to be used with many
different promoters including ubiquitous, tissue specific, and
regulatable promoters. In addition, adenoviruses can be
administered by intravenous injection, because the viruses are
stable in the bloodstream.
[0295] Using adenovirus vectors where portions of the adenovirus
genome are deleted, inserts are incorporated into the viral DNA by
direct ligation or by homologous recombination with a
co-transfected plasmid. In an exemplary system, the essential E1
gene is deleted from the viral vector, and the virus will not
replicate unless the E1 gene is provided by the host cell. When
intravenously administered to intact animals, adenovirus primarily
targets the liver. Although an adenoviral delivery system with an
E1 gene deletion cannot replicate in the host cells, the host's
tissue will express and process an encoded heterologous protein.
Host cells will also secrete the heterologous protein if the
corresponding gene includes a secretory signal sequence. Secreted
proteins will enter the circulation from tissue that expresses the
heterologous gene (e.g., the highly vascularized liver).
[0296] Moreover, adenoviral vectors containing various deletions of
viral genes can be used to reduce or eliminate immune responses to
the vector. Such adenoviruses are E1-deleted, and in addition,
contain deletions of E2A or E4 (Lusky et al., J. Virol. 72:2022
(1998); Raper et al., Human Gene Therapy 9:671 (1998)). The
deletion of E2b has also been reported to reduce immune responses
(Amalfitano et al., J. Virol. 72:926 (1998)). By deleting the
entire adenovirus genome, very large inserts of heterologous DNA
can be accommodated. Generation of so called "gutless"
adenoviruses, where all viral genes are deleted, are particularly
advantageous for insertion of large inserts of heterologous DNA
(for a review, see Yeh. and Perricaudet, FASEB J. 11:615
(1997)).
[0297] High titer stocks of recombinant viruses capable of
expressing a therapeutic gene can be obtained from infected
mammalian cells using standard methods. For example, recombinant
HSV can be prepared in Vero cells, as described by Brandt et al.,
J. Gen. Virol. 72:2043 (1991), Herold et al., J. Gen. Virol.
75:1211 (1994), Visalli and Brandt, Virology 185:419 (1991), Grau
et al., Invest. Ophthalmol. Vis. Sci. 30:2474 (1989), Brandt et
al., J. Virol. Meth. 36:209 (1992), and by Brown and MacLean
(eds.), HSV Virus Protocols (Humana Press 1997).
[0298] Alternatively, an expression vector comprising a zupar1 gene
can be introduced into a subject's cells by lipofection in vivo
using liposomes. Synthetic cationic lipids can be used to prepare
liposomes for in vivo transfection of a gene encoding a marker
(Felgner et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey
et al., Proc. Nat'l Acad. Sci. USA 85:8027 (1988)). The use of
lipofection to introduce exogenous genes into specific organs in
vivo has certain practical advantages. Liposomes can be used to
direct transfection to particular cell types, which is particularly
advantageous in a tissue with cellular heterogeneity, such as the
pancreas, liver, kidney, and brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting. Targeted
peptides (e.g., hormones or neurotransmitters), proteins such as
antibodies, or non-peptide molecules can be coupled to liposomes
chemically.
[0299] Electroporation is another alternative mode of
administration of a zupar1 nucleic acid molecules. For example,
Aihara and Miyazaki, Nature Biotechnology 16:867 (1998), have
demonstrated the use of in vivo electroporation for gene transfer
into muscle.
[0300] In an alternative approach to gene therapy, a therapeutic
gene may encode a zupar1 anti-sense RNA that inhibits the
expression of zupar1. Methods of preparing anti-sense constructs
are known to those in the art. See, for example, Erickson et al.,
Dev. Genet. 14:274 (1993) [transgenic mice], Augustine et al., Dev.
Genet. 14:500 (1993) [murine whole embryo culture], and Olson and
Gibo, Exp. Cell Res. 241:134 (1998) [cultured cells]. Suitable
sequences for zupar1 anti-sense molecules can be derived from the
nucleotide sequences of zupar1 disclosed herein.
[0301] Alternatively, an expression vector can be constructed in
which a regulatory element is operably linked to a nucleotide
sequence that encodes a ribozyme. Ribozymes can be designed to
express endonuclease activity that is directed to a certain target
sequence in a mRNA molecule (see, for example, Draper and Macejak,
U.S. Pat. No. 5,496,698, McSwiggen, U.S. Pat. No. 5,525,468,
Chowrira and McSwiggen, U.S. Pat. No. 5,631,359, and Robertson and
Goldberg, U.S. Pat. No. 5,225,337). In the context of the present
invention, ribozymes include nucleotide sequences that bind with
zupar1 mRNA.
[0302] In another approach, expression vectors can be constructed
in which a regulatory element directs the production of RNA
transcripts capable of promoting RNase P-mediated cleavage of mRNA
molecules that encode a zupar1 gene. According to this approach, an
external guide sequence can be constructed for directing the
endogenous ribozyme, RNase P, to a particular species of
intracellular mRNA, which is subsequently cleaved by the cellular
ribozyme (see, for example, Altman et al., U.S. Pat. No. 5,168,053,
Yuan et al., Science 263:1269 (1994), Pace et al., international
publication No. WO 96/18733, George et al., international
publication No. WO 96/21731, and Werner et al., international
publication No. WO 97/33991). Preferably, the external guide
sequence comprises a ten to fifteen nucleotide sequence
complementary to zupar1 mRNA, and a 3'-NCCA nucleotide sequence,
wherein N is preferably a purine. The external guide sequence
transcripts bind to the targeted mRNA species by the formation of
base pairs between the mRNA and the complementary external guide
sequences, thus promoting cleavage of mRNA by RNase P at the
nucleotide located at the 5'-side of the base-paired region.
[0303] In general, the dosage of a composition comprising a
therapeutic vector having a zupar1 nucleotide acid sequence, such
as a recombinant virus, will vary depending upon such factors as
the subject's age, weight, height, sex, general medical condition
and previous medical history. Suitable routes of administration of
therapeutic vectors include intravenous injection, intraarterial
injection, intraperitoneal injection, intramuscular injection,
intratumoral injection, and injection into a cavity that contains a
tumor.
[0304] A composition comprising viral vectors, non-viral vectors,
or a combination of viral and non-viral vectors of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby vectors or viruses
are combined in a mixture with a pharmaceutically acceptable
carrier. As noted above, a composition, such as phosphate-buffered
saline is said to be a "pharmaceutically acceptable carrier" if its
administration can be tolerated by a recipient subject. Other
suitable carriers are well-known to those in the art (see, for
example, Remington's Pharmaceutical Sciences, 19th Ed. (Mack
Publishing Co. 1995), and Gilman's the Pharmacological Basis of
Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985)).
[0305] For purposes of therapy, a therapeutic gene expression
vector, or a recombinant virus comprising such a vector, and a
pharmaceutically acceptable carrier are administered to a subject
in a therapeutically effective amount. A combination of an
expression vector (or virus) and a pharmaceutically acceptable
carrier is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results in
a detectable change in the physiology of a recipient subject.
[0306] When the subject treated with a therapeutic gene expression
vector or a recombinant virus is a human, then the therapy is
preferably somatic cell gene therapy. That is, the preferred
treatment of a human with a therapeutic gene expression vector or a
recombinant virus does not entail introducing into cells a nucleic
acid molecule that can form part of a human germ line and be passed
onto successive generations (i.e., human germ line gene
therapy).
EXAMPLES
Example 1
[0307] zupar1 is produced in E. coli using a His.sub.6 tag/maltose
binding protein (MBP) double affinity fusion system as generally
disclosed by Pryor and Leiting, Prot. Expr. Pur. 10:309-319, 1997.
A thrombin cleavage site is placed at the junction between the
affinity tag and zupar1 sequences.
[0308] The fusion construct is assembled in the vector pTAP98,
which comprises sequences for replication and selection in E. coli
and yeast, the E. coli tac promoter, and a unique Smal site just
downstream of the MBP-His.sub.6-thrombin site coding sequences. The
zupar1 cDNA is amplified by PCR using primers each comprising 40 bp
of sequence homologous to vector sequence and 25 bp of sequence
that anneals to the zupar1 cDNA. The reaction is run using Taq DNA
polymerase (Boehringer Mannheim, Indianapolis, Ind.) for 30 cycles
of 94.degree. C., 30 seconds; 60.degree. C., 60 seconds; and
72.degree. C., 60 seconds. One microgram of the resulting fragment
is mixed with 100 ng of SmaI-cut pTAP98, and the mixture is
transformed into yeast to assemble the vector by homologous
recombination (Oldenburg et al., Nucl. Acids. Res. 25:451-452,
1997). Ura.sup.+ transformants are selected.
[0309] Plasmid DNA is prepared from yeast transformants and
transformed into E. coli MC1061. Pooled plasmid DNA is then
prepared from the MC1061 transformants by the miniprep method after
scraping an entire plate. Plasmid DNA is analyzed by restriction
digestion.
[0310] E. coli strain BL21 is used for expression of zupar1. Cells
are transformed by electroporation and grown on minimal glucose
plates containing casamino acids and ampicillin.
[0311] Protein expression is analyzed by gel electrophoresis. Cells
are grown in liquid glucose media containing casamino acids and
ampicillin. After one hour at 37.degree. C., IPTG is added to a
final concentration of 1 mM, and the cells are grown for an
additional 2-3 hours at 37.degree. C. Cells are disrupted using
glass beads, and extracts are prepared.
Example 2
[0312] Larger scale cultures of zupar1 transformants are prepared
by the method of Pryor and Leiting (ibid.). 100-ml cultures in
minimal glucose media containing casamino acids and 100 .mu.g/ml
ampicillin are grown at 37.degree. C. in 500-ml baffled flasks to
OD.sub.600.apprxeq.0.5. Cells are harvested by centrifugation and
resuspended in 100 ml of the same media at room temperature. After
15 minutes, IPTG is added to 0.5 mM, and cultures are incubated at
room temperature (ca. 22.5.degree. C.) for 16 to 20 hours with
shaking at 125 rpm. The culture is harvested by centrifugation, and
cell pellets are stored at -70.degree. C.
Example 3
[0313] For larger-scale protein preparation, 500-ml cultures of E.
coli BL21 expressing the zupar1-MBP-His.sub.6 fusion protein are
prepared essentially as disclosed in Example 2. Cell pellets are
resuspended in 100 ml of binding buffer (20 mM Tris, pH 7.58, 100
mM NaCl, 20 mM NaH.sub.2PO.sub.4, 0.4 mM
4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride
[Pefabloc.RTM. SC; Boehringer-Mannheim], 2 .mu.g/ml Leupeptin, 2
.mu.g/ml Aprotinin). The cells are lysed in a French press at
30,000 psi, and the lysate is centrifuged at 18,000.times.g for 45
minutes at 4.degree. C. to clarify it. Protein concentration is
estimated by gel electrophoresis with a BSA standard.
[0314] The recombinant zupar1 fusion protein is purified from the
lysate by affinity chromatography. Immobilized cobalt resin
(Talon.RTM. resin; Clontech Laboratories, Inc., Palo Alto, Calif.)
is equilibrated in binding buffer. One ml of packed resin per 50 mg
protein is combined with the clarified supernatant in a tube, and
the tube is capped and sealed, then placed on a rocker overnight at
4.degree. C. The resin is then pelleted by centrifugation at
4.degree. C. and washed three times with binding buffer. Protein is
eluted with binding buffer containing 0.2 M imidazole. The resin
and elution buffer are mixed for at least one hour at 4.degree. C.,
the resin is pelleted, and the supernatant is removed. An aliquot
is analyzed by gel electrophoresis, and concentration is estimated.
Amylose resin is equilibrated in amylose binding buffer (20 mM
Tris-HCl, pH 7.0, 100 mM NaCl, 10 mM EDTA) and combined with the
supernatant from the Talon resin at a ratio of 2 mg fusion protein
per ml of resin. Binding and washing steps are carried out as
disclosed above. Protein is eluted with amylose binding buffer
containing 10 mM maltose using as small a volume as possible to
minimize the need for subsequent concentration. The eluted protein
is analyzed by gel electrophoresis and staining with Coomassie blue
using a BSA standard, and by Western blotting using an anti-MBP
antibody.
Example 4
[0315] An expression plasmid containing all or part of a
polynucleotide encoding zuapr1 is constructed via homologous
recombination. A zupar1 coding 20 sequence comprising the ORF with
5' and 3' ends corresponding to the vector sequences flanking the
insertion point is prepared by PCR. The primers for PCR each
include from 5' to 3' end: 40 bp of flanking sequence from the
vector and 17 bp corresponding to the amino or carboxyl termini
from the open reading frame of zupar1.
[0316] Ten .mu.l of the 100 .mu.l PCR reaction mixture is run on a
0.8% low-melting-temperature agarose (SeaPlaque GTG.RTM.; FMC
BioProducts, Rockland, Me.) gel with 1.times.TBE buffer for
analysis. The remaining 90 .mu.l of the reaction mixture is
precipitated with the addition of 5 .mu.l 1 M NaCl and 250 .mu.l of
absolute ethanol. The plasmid pZMP6, which has been cut with SmaI,
is used for recombination with the PCR fragment. Plasmid pZMP6 is a
mammalian expression vector containing an expression cassette
having the cytomegalovirus immediate early promoter, multiple
restriction sites for insertion of coding sequences, a stop codon,
and a human growth hormone terminator; an E. coli origin of
replication; a mammalian selectable marker expression unit
comprising an SV40 promoter, enhancer and origin of replication, a
DHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequences
required for selection and replication in S. cerevisiae. It was
constructed from pZP9 (deposited at the American Type Culture
Collection, 10801 University Boulevard, Manassas, Va. 20110-2209,
under Accession No. 98668) with the yeast genetic elements taken
from pRS316 (available from the American Type Culture Collection,
10801 University Boulevard, Manassas, Va., under Accession No.
77145), an internal ribosome entry site (IRES) element from
poliovirus, and the extracellular domain of CD8 truncated at the
C-terminal end of the transmembrane domain.
[0317] One hundred microliters of competent yeast (S. cerevisiae)
cells are independently combined with 10 .mu.l of the various DNA
mixtures from above and transferred to a 0.2-cm electroporation
cuvette. The yeast/DNA mixtures are electropulsed using power
supply (BioRad Laboratories, Hercules, Calif.) settings of 0.75 kV
(5 kV/cm), .infin. ohms, 25 .mu.F. To each cuvette is added 600
.mu.l of 1.2 M sorbitol, and the yeast is plated in two 300-.mu.l
aliquots onto two URA-D plates (1.8% agar in 2% D-glucose, 0.67%
yeast nitrogen base without amino acids, 0.056% -Ura-Trp-Thr powder
[made by combining 4.0 g L-adenine, 3.0 g L-arginine, 5.0 g
L-aspartic acid, 2.0 g L-histidine, 6.0 g L-isoleucine, 8.0 g
L-leucine, 4.0 g L-lysine, 2.0 g L-methionine, 6.0 g
L-phenylalanine, 5.0 g L-serine, 5.0 g L-tyrosine, and 6.0 g
L-valine], and 0.5% 200.times.tryptophan, threonine solution [3.0%
L-threonine, 0.8% L-tryptophan in H.sub.2O]) and incubated at
30.degree. C. After about 48 hours, the Ura.sup.+ yeast
transformants from a single plate are resuspended in 1 ml H.sub.2O
and spun briefly to pellet the yeast cells. The cell pellet is
resuspended in 1 m1 of lysis buffer (2% Triton X-100, 1% SDS, 100
mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundred microliters
of the lysis mixture is added to an Eppendorf tube containing 300
.mu.l acid-washed glass beads and 200 .mu.l phenol-chloroform,
vortexed for 1 minute intervals two or three times, and spun for 5
minutes in an Eppendorf centrifuge at maximum speed. Three hundred
microliters of the aqueous phase is transferred to a fresh tube,
and the DNA is precipitated with 600 .mu.l ethanol (EtOH), followed
by centrifugation for 10 minutes at 4.degree. C. The DNA pellet is
resuspended in 10 l H.sub.2O.
[0318] Transformation of electrocompetent E. coli host cells
(Electromax DH10B.TM. cells; obtained from Life Technologies, Inc.,
Gaithersburg, Md.) is done with 0.5-2 ml yeast DNA prep and 40
.mu.l of cells. The cells are electropulsed at 1.7 kV, 25 .mu.F,
and 400 ohms. Following electroporation, 1 mil SOC (2% Bacto.TM.
Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM
NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4, 20 mM
glucose) is plated in 250-.mu.l aliquots on four LB AMP plates (LB
broth (Lennox), 1.8% Bacto.TM. Agar (Difco), 100 mg/L
Ampicillin).
[0319] Individual clones harboring the correct expression construct
for zupar1 are identified by restriction digest to verify the
presence of the zupar1 insert and to confirm that the various DNA
sequences have been joined correctly to one another. The inserts of
positive clones are subjected to sequence analysis. Larger scale
plasmid DNA is isolated using a commercially available kit (QIAGEN
Plasmid Maxi Kit, Qiagen, Valencia, Calif.) according to
manufacturer's instructions. The correct construct is designated
pZMP6/ zupar1.
[0320] Recombinant protein is produced in BHK cells transfected
with pZMP6/ zupar1. BHK 570 cells (ATCC CRL-10314) are plated in
10-cm tissue culture dishes and allowed to grow to approximately 50
to 70% confluence overnight at 37.degree. C., 5% CO.sub.2, in
DMEM/FBS media (DMEM, Gibco/BRL High Glucose; Life Technologies),
5% fetal bovine serum (Hyclone, Logan, Utah), 1 mM L-glutamine (JRH
Biosciences, Lenexa, Kans.), 1 mM sodium pyruvate (Life
Technologies). The cells are then transfected with pZMP6/zupar1 by
liposome-mediated transfection using a 3:1 (w/w) liposome
formulation of the polycationic lipid
2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanminiu-
m-trifluoroacetate and the neutral lipid dioleoyl
phosphatidylethanolamine in membrane-filtered water
(Lipofectamine.TM. Reagent; Life Technologies, Gaithersburg, Md.),
in serum free (SF) media (DMEM supplemented with 10 mg/ml
transferrin, 5 mg/m1 insulin, 2 mg/ml fetuin, 1% L-glutamine and 1%
sodium pyruvate). The plasmid is diluted into 15-ml tubes to a
total final volume of 640 .mu.l with SF media. 35 .mu.l of the
lipid mixture is mixed with 605 .mu.l of SF medium, and the
resulting mixture is allowed to incubate approximately 30 minutes
at room temperature. Five milliliters of SF media is then added to
the DNA:lipid mixture. The cells are rinsed once with 5 ml of SF
media, aspirated, and the DNA:lipid mixture is added. The cells are
incubated at 37.degree. C. for five hours, then 6.4 ml of DMEM/10%
FBS, 1% PSN media is added to each plate. The plates are incubated
at 37.degree. C. overnight, and the DNA:lipid mixture is replaced
with fresh 5% FBS/DMEM media the next day. On day 5
post-transfection, the cells are split into T-162 flasks in
selection medium (DMEM +5% FBS, 1% L-Gln, 1% NaPyr, 1 .mu.M
methotrexate). Approximately 10 days post-transfection, two 150-mm
culture dishes of methotrexate-resistant colonies from each
transfection are trypsinized, and the cells are pooled and plated
into a T-162 flask and transferred to large-scale culture.
[0321] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
11 1 375 DNA Homo sapiens CDS (1)...(372) 1 atg gtc ctg tgc tgg ctg
ctg ctt ctg gtg atg gct ctg ccc cca ggc 48 Met Val Leu Cys Trp Leu
Leu Leu Leu Val Met Ala Leu Pro Pro Gly 1 5 10 15 acg acg ggc gtc
aag gac tgc gtc ttc tgt gag ctc acc gac tcc atg 96 Thr Thr Gly Val
Lys Asp Cys Val Phe Cys Glu Leu Thr Asp Ser Met 20 25 30 cag tgt
cct ggt acc tac atg cac tgt ggc gat gac gag gac tgc ttc 144 Gln Cys
Pro Gly Thr Tyr Met His Cys Gly Asp Asp Glu Asp Cys Phe 35 40 45
aca ggc cac ggg gtc gcc ccg ggc act ggt ccg gtc atc aac aaa ggc 192
Thr Gly His Gly Val Ala Pro Gly Thr Gly Pro Val Ile Asn Lys Gly 50
55 60 tgc ctg cga gcc acc agc tgc ggc ctt gag gaa ccc gtc agc tac
agg 240 Cys Leu Arg Ala Thr Ser Cys Gly Leu Glu Glu Pro Val Ser Tyr
Arg 65 70 75 80 ggc gtc acc tac agc ctc acc acc aac tgc tgc acc ggc
cgc ctg tgt 288 Gly Val Thr Tyr Ser Leu Thr Thr Asn Cys Cys Thr Gly
Arg Leu Cys 85 90 95 aac aga gcc ccg agc agc cag aca gtg ggg gcc
acc acc agc ctg gca 336 Asn Arg Ala Pro Ser Ser Gln Thr Val Gly Ala
Thr Thr Ser Leu Ala 100 105 110 ctg ggg ctg ggt atg ctg ctt cct cca
cgt ttg ctg tga 375 Leu Gly Leu Gly Met Leu Leu Pro Pro Arg Leu Leu
115 120 2 124 PRT Homo sapiens 2 Met Val Leu Cys Trp Leu Leu Leu
Leu Val Met Ala Leu Pro Pro Gly 1 5 10 15 Thr Thr Gly Val Lys Asp
Cys Val Phe Cys Glu Leu Thr Asp Ser Met 20 25 30 Gln Cys Pro Gly
Thr Tyr Met His Cys Gly Asp Asp Glu Asp Cys Phe 35 40 45 Thr Gly
His Gly Val Ala Pro Gly Thr Gly Pro Val Ile Asn Lys Gly 50 55 60
Cys Leu Arg Ala Thr Ser Cys Gly Leu Glu Glu Pro Val Ser Tyr Arg 65
70 75 80 Gly Val Thr Tyr Ser Leu Thr Thr Asn Cys Cys Thr Gly Arg
Leu Cys 85 90 95 Asn Arg Ala Pro Ser Ser Gln Thr Val Gly Ala Thr
Thr Ser Leu Ala 100 105 110 Leu Gly Leu Gly Met Leu Leu Pro Pro Arg
Leu Leu 115 120 3 372 DNA Artificial Sequence This degenerate
nucleotide sequence encodes the amino acid sequence of SEQ ID NO2.
3 atggtnytnt gytggytnyt nytnytngtn atggcnytnc cnccnggnac nacnggngtn
60 aargaytgyg tnttytgyga rytnacngay wsnatgcart gyccnggnac
ntayatgcay 120 tgyggngayg aygargaytg yttyacnggn cayggngtng
cnccnggnac nggnccngtn 180 athaayaarg gntgyytnmg ngcnacnwsn
tgyggnytng argarccngt nwsntaymgn 240 ggngtnacnt aywsnytnac
nacnaaytgy tgyacnggnm gnytntgyaa ymgngcnccn 300 wsnwsncara
cngtnggngc nacnacnwsn ytngcnytng gnytnggnat gytnytnccn 360
ccnmgnytny tn 372 4 16 PRT Artificial Sequence Polypeptide linker.
4 Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1
5 10 15 5 106 PRT Homo sapiens 5 Gly Val Lys Asp Cys Val Phe Cys
Glu Leu Thr Asp Ser Met Gln Cys 1 5 10 15 Pro Gly Thr Tyr Met His
Cys Gly Asp Asp Glu Asp Cys Phe Thr Gly 20 25 30 His Gly Val Ala
Pro Gly Thr Gly Pro Val Ile Asn Lys Gly Cys Leu 35 40 45 Arg Ala
Thr Ser Cys Gly Leu Glu Glu Pro Val Ser Tyr Arg Gly Val 50 55 60
Thr Tyr Ser Leu Thr Thr Asn Cys Cys Thr Gly Arg Leu Cys Asn Arg 65
70 75 80 Ala Pro Ser Ser Gln Thr Val Gly Ala Thr Thr Ser Leu Ala
Leu Gly 85 90 95 Leu Gly Met Leu Leu Pro Pro Arg Leu Leu 100 105 6
92 PRT Homo sapiens 6 Gly Val Lys Asp Cys Val Phe Cys Glu Leu Thr
Asp Ser Met Gln Cys 1 5 10 15 Pro Gly Thr Tyr Met His Cys Gly Asp
Asp Glu Asp Cys Phe Thr Gly 20 25 30 His Gly Val Ala Pro Gly Thr
Gly Pro Val Ile Asn Lys Gly Cys Leu 35 40 45 Arg Ala Thr Ser Cys
Gly Leu Glu Glu Pro Val Ser Tyr Arg Gly Val 50 55 60 Thr Tyr Ser
Leu Thr Thr Asn Cys Cys Thr Gly Arg Leu Cys Asn Arg 65 70 75 80 Ala
Pro Ser Ser Gln Thr Val Gly Ala Thr Thr Ser 85 90 7 79 PRT Homo
sapiens 7 Gly Val Lys Asp Cys Val Phe Cys Glu Leu Thr Asp Ser Met
Gln Cys 1 5 10 15 Pro Gly Thr Tyr Met His Cys Gly Asp Asp Glu Asp
Cys Phe Thr Gly 20 25 30 His Gly Val Ala Pro Gly Thr Gly Pro Val
Ile Asn Lys Gly Cys Leu 35 40 45 Arg Ala Thr Ser Cys Gly Leu Glu
Glu Pro Val Ser Tyr Arg Gly Val 50 55 60 Thr Tyr Ser Leu Thr Thr
Asn Cys Cys Thr Gly Arg Leu Cys Asn 65 70 75 8 105 PRT Homo sapiens
8 Val Lys Asp Cys Val Phe Cys Glu Leu Thr Asp Ser Met Gln Cys Pro 1
5 10 15 Gly Thr Tyr Met His Cys Gly Asp Asp Glu Asp Cys Phe Thr Gly
His 20 25 30 Gly Val Ala Pro Gly Thr Gly Pro Val Ile Asn Lys Gly
Cys Leu Arg 35 40 45 Ala Thr Ser Cys Gly Leu Glu Glu Pro Val Ser
Tyr Arg Gly Val Thr 50 55 60 Tyr Ser Leu Thr Thr Asn Cys Cys Thr
Gly Arg Leu Cys Asn Arg Ala 65 70 75 80 Pro Ser Ser Gln Thr Val Gly
Ala Thr Thr Ser Leu Ala Leu Gly Leu 85 90 95 Gly Met Leu Leu Pro
Pro Arg Leu Leu 100 105 9 91 PRT Homo sapiens 9 Val Lys Asp Cys Val
Phe Cys Glu Leu Thr Asp Ser Met Gln Cys Pro 1 5 10 15 Gly Thr Tyr
Met His Cys Gly Asp Asp Glu Asp Cys Phe Thr Gly His 20 25 30 Gly
Val Ala Pro Gly Thr Gly Pro Val Ile Asn Lys Gly Cys Leu Arg 35 40
45 Ala Thr Ser Cys Gly Leu Glu Glu Pro Val Ser Tyr Arg Gly Val Thr
50 55 60 Tyr Ser Leu Thr Thr Asn Cys Cys Thr Gly Arg Leu Cys Asn
Arg Ala 65 70 75 80 Pro Ser Ser Gln Thr Val Gly Ala Thr Thr Ser 85
90 10 78 PRT Homo sapiens 10 Val Lys Asp Cys Val Phe Cys Glu Leu
Thr Asp Ser Met Gln Cys Pro 1 5 10 15 Gly Thr Tyr Met His Cys Gly
Asp Asp Glu Asp Cys Phe Thr Gly His 20 25 30 Gly Val Ala Pro Gly
Thr Gly Pro Val Ile Asn Lys Gly Cys Leu Arg 35 40 45 Ala Thr Ser
Cys Gly Leu Glu Glu Pro Val Ser Tyr Arg Gly Val Thr 50 55 60 Tyr
Ser Leu Thr Thr Asn Cys Cys Thr Gly Arg Leu Cys Asn 65 70 75 11 701
DNA Mus musculus CDS (233)...(613) 11 agagttccat ggcccaaagg
cttgtgagca ggggtgctgg cccatctcca acccgaggac 60 tgaagagctt
ctgaaaagga cacgttgtat cgggaacccg aaacactcca actgctgacc 120
cccaaggcca ggacgagggg caggaggtgt tccaggaggt cacactcggc catccaggag
180 gtcaaggaag cccccggcct ggcttgggtg ctatccacgc cgccaggcca cc atg
gtc 238 Met Val 1 ctt ggc tgg cca ctg ctt ctg gtg ttg gtt ctt tgc
cca ggt gtg aca 286 Leu Gly Trp Pro Leu Leu Leu Val Leu Val Leu Cys
Pro Gly Val Thr 5 10 15 ggc atc aag gac tgc gtc ttc tgt gag ctg act
gac tct gct cgg tgc 334 Gly Ile Lys Asp Cys Val Phe Cys Glu Leu Thr
Asp Ser Ala Arg Cys 20 25 30 cct ggc aca cac atg cgc tgt ggg gat
gac gaa gat tgc ttc aca ggc 382 Pro Gly Thr His Met Arg Cys Gly Asp
Asp Glu Asp Cys Phe Thr Gly 35 40 45 50 cac gga gta gcc cag ggt gtg
ggg ccc atc atc aac aga ggc tgc gtg 430 His Gly Val Ala Gln Gly Val
Gly Pro Ile Ile Asn Arg Gly Cys Val 55 60 65 cac tcc acc agc tgt
ggc cgc gag gaa ccc atc agc tac atg ggc ctc 478 His Ser Thr Ser Cys
Gly Arg Glu Glu Pro Ile Ser Tyr Met Gly Leu 70 75 80 aca tac agt
ctc acc acc acc tgc tgt tct ggc cac ctt tgc aat aag 526 Thr Tyr Ser
Leu Thr Thr Thr Cys Cys Ser Gly His Leu Cys Asn Lys 85 90 95 ggc
act ggc ctt tcc aca ggg gct acc agc ctg tca ctg ggt ctg cag 574 Gly
Thr Gly Leu Ser Thr Gly Ala Thr Ser Leu Ser Leu Gly Leu Gln 100 105
110 ctg ctc ctg ggc ctg ttg ctg ctg ctt caa tac tgg ctg tgacccacaa
623 Leu Leu Leu Gly Leu Leu Leu Leu Leu Gln Tyr Trp Leu 115 120 125
gaactgcagg gcctagggct gtgcccccgg cttccgcctt catccctttc ccccattaaa
683 tggccagagg tgcagtct 701
* * * * *