U.S. patent application number 10/777578 was filed with the patent office on 2004-07-29 for novel secreted proteins.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Presnell, Scott R., Sheppard, Paul O., Taft, David W..
Application Number | 20040147722 10/777578 |
Document ID | / |
Family ID | 22803010 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040147722 |
Kind Code |
A1 |
Sheppard, Paul O. ; et
al. |
July 29, 2004 |
Novel secreted proteins
Abstract
The present invention provides to polynucleotides and secreted
proteins encoded by the polynucleotides. The proteins include and
cytokines, growth factors, include fusion proteins operably linked
to a second polypeptide. The invention further provides therapeutic
and diagnostic methods utilizing the polynucleotides, polypeptides,
and antagonists of the polypeptides.
Inventors: |
Sheppard, Paul O.; (Granite
Falls, WA) ; Presnell, Scott R.; (Tacoma, WA)
; Taft, David W.; (Seattle, WA) |
Correspondence
Address: |
Robyn Adams
Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
22803010 |
Appl. No.: |
10/777578 |
Filed: |
February 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10777578 |
Feb 12, 2004 |
|
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09895836 |
Jun 29, 2001 |
|
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60215446 |
Jun 30, 2000 |
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Current U.S.
Class: |
530/350 ;
435/320.1; 435/325; 435/69.1; 530/351; 530/388.1; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/47 20130101; C07K 14/52 20130101 |
Class at
Publication: |
530/350 ;
530/388.1; 435/069.1; 435/320.1; 435/325; 536/023.5; 530/351 |
International
Class: |
C07H 021/04; C07K
014/52; C07K 014/475; C07K 016/24 |
Claims
We claim:
1. An isolated polypeptide comprising residues 23 to 150 as shown
in SEQ ID NO:2.
2. The isolated polypeptide of claim 1 wherein the polypeptide
further comprises residues 1 to 150 as shown in SEQ ID NO:2.
3. An isolated polynucleotide comprising a sequence of nucleotides,
wherein the sequence encodes the isolated polypeptide of claim
1.
4. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment having the
isolated polynucleotide of claim 3; and a transcription
terminator.
5. A cultured cell comprising the expression vector of claim 4.
6. A method of producing a polypeptide comprising culturing the
cell of claim 5 under conditions whereby said sequence of
nucleotides is expressed, and recovering said polypeptide.
7. A polypeptide produced by the method of claim 6.
8. An isolated polynucleotide comprising the polynucleotide
sequence as shown in SEQ ID NO: 1.
9. An antibody that specifically binds to the isolated protein of
claim 1.
10. An isolated polypeptide comprising residues 19 to 117 as shown
in SEQ ID NO:5.
11. The isolated polypeptide of claim 10 wherein the polypeptide
further comprises residues 1 to 117 as shown in SEQ ID NO:5.
12. An isolated polynucleotide comprising a sequence of
nucleotides, wherein the sequence encodes the isolated polypeptide
of claim 10.
13. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment having the
isolated polynucleotide of claim 12; and a transcription
terminator.
14. A cultured cell comprising the expression vector of claim
13.
15. A method of producing a polypeptide comprising culturing the
cell of claim 14 under conditions whereby said sequence of
nucleotides is expressed, and recovering said polypeptide.
16. A polypeptide produced by the method of claim 15
17. An isolated polynucleotide comprising the polynucleotide
sequence as shown in SEQ ID NO:4.
18. An antibody that specifically binds to the isolated protein of
claim 10.
19. An isolated polypeptide comprising residues 20 to 185 as shown
in SEQ ID NO:8.
20. The isolated polypeptide of claim 19 wherein the polypeptide
further comprises residues 1 to 185 as shown in SEQ ID NO:8.
21. An isolated polynucleotide comprising a sequence of
nucleotides, wherein the sequence encodes the isolated polypeptide
of claim 19.
22. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment having the
isolated polynucleotide of claim 21; and a transcription
terminator.
23. A cultured cell comprising the expression vector of claim
22.
24. A method of producing a polypeptide comprising culturing the
cell of claim 23 under conditions whereby said sequence of
nucleotides is expressed, and recovering said polypeptide.
25. A polypeptide produced by the method of claim 24.
26. An isolated polynucleotide comprising the polynucleotide
sequence as shown in SEQ ID NO:7.
27. An antibody that specifically binds to the isolated protein of
claim 19.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/895,836, filed Jun. 29, 2001, which claims the benefit of
U.S. Provisional Application Serial No. 60/215,446 filed Jun. 30,
2000, both of which are herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Within the field of genetic engineering, polynucleotides
encoding proteins of interest have been identified and cloned by
methods that require a detailed knowledge of the structure and/or
function of the polynucleotide or the encoded protein. These
methods include hybridization screening, polymerase chain redaction
(PCR), and expression cloning.
[0003] With the more recent advent of large DNA sequence databases
and the accompanying data analysis tools, identification of genes
of interest is possible through the analysis of raw sequence data.
Databases can be "mined" to locate sequences that resemble (are
"homologous to") sequences of known function. Alignment of similar
sequences can be used to place novel sequences within families of
structurally similar sequences. These analytical tools can be
combined with structural information obtained from, for example,
X-ray crystallography to predict the higher order structure of a
novel polypeptide. These analyses also facilitate prediction of
polypeptide function. These recent technological advances have
greatly increased the pace of gene discovery.
[0004] Genetic engineering has made available a number of genes and
proteins of pharmaceutical or other economic importance. Such
proteins include, for example, tissue plasminogen activator (t-PA)
(U.S. Pat. No. 4,766,075), coagulation factor VII (U.S. Pat. No.
4,784,950), erythropoietin (U.S. Pat. No. 4,703,008), platelet
derived growth factor (U.S. Pat. No. 4,889,919), and various
industrial enzymes (e.g., U.S. Pat. Nos. 5,965,384; 5,942,431; and
5,922,586).
[0005] Although estimates vary as to the amount of the human genome
that has been identified to date, there remains a need in the art
for further characterization of the human genome and the proteins
encoded thereby. Previously unknown genes and proteins will be
useful in the treatment and/or prevention of many human diseases,
included diseases that have heretofore been refractory to
treatment.
SUMMARY OF THE INVENTION
[0006] Within one aspect the invention provides an isolated
polypeptide comprising residues 23 to 150 as shown in SEQ ID NO:2.
Within an embodiment, the polypeptide further comprises residues 1
to 150 as shown in SEQ ID NO:2.
[0007] Within another aspect the invention provides an isolated
polynucleotide comprising a sequence of nucleotides, wherein the
sequence encodes an isolated comprising residues 23 to 150 as shown
in SEQ ID NO:2. Within an embodiment, is provided an expression
vector comprising the following operably linked elements: a
transcription promoter; a DNA segment having the isolated
polynucleotide; and a transcription terminator. Within another
embodiment, the invention provides a cultured cell comprising the
expression vector. Within another embodiment is provided a method
of producing a polypeptide comprising culturing the cell under
conditions whereby said sequence of nucleotides is expressed, and
recovering said polypeptide. Within another embodiment the
polypeptide produced by the method is provided
[0008] Within another aspect the invention provides an isolated
polynucleotide comprising the polynucleotide sequence as shown in
SEQ ID NO: 1.
[0009] Within another aspect the invention provides an antibody
that specifically binds to the isolated protein comprising residues
23 to 150 as shown in SEQ ID NO:2.
[0010] Within one aspect the invention provides an isolated
polypeptide comprising residues 19 to 117 as shown in SEQ ID NO:5.
Within an embodiment, the polypeptide further comprises residues 1
to 117 as shown in SEQ ID NO:5.
[0011] Within another aspect the invention provides an isolated
polynucleotide comprising a sequence of nucleotides, wherein the
sequence encodes an isolated comprising residues 19 to 117 as shown
in SEQ. ID NO:5. Within an embodiment, is provided an expression
vector comprising the following operably linked elements: a
transcription promoter; a DNA segment having the isolated
polynucleotide; and a transcription terminator. Within another
embodiment, the invention provides a cultured cell comprising the
expression vector. Within another embodiment is provided a method
of producing a polypeptide comprising culturing the cell under
conditions whereby said sequence of nucleotides is expressed, and
recovering said polypeptide. Within another embodiment the
polypeptide produced by the method is provided
[0012] Within another aspect the invention provides an isolated
polynucleotide comprising the polynucleotide sequence as shown in
SEQ ID NO:4.
[0013] Within another aspect the invention provides an antibody
that specifically binds to the isolated protein comprising residues
19 to 117 as shown in SEQ ID NO:5.
[0014] Within one aspect the invention provides an isolated
polypeptide comprising residues 20 to 185 as shown in SEQ ID NO:8.
Within an embodiment, the polypeptide further comprises residues 1
to 185 as shown in SEQ ID NO:8.
[0015] Within another aspect the invention provides an isolated
polynucleotide comprising a sequence of nucleotides, wherein the
sequence encodes an isolated comprising residues 20 to 185 as shown
in SEQ ID NO:8. Within an embodiment, is provided an expression
vector comprising the following operably linked elements: a
transcription promoter; a DNA segment having the isolated
polynucleotide; and a transcription terminator. Within another
embodiment, the invention provides a cultured cell comprising the
expression vector. Within another embodiment is provided; a method
of producing a polypeptide comprising culturing the cell under
conditions whereby said sequence of nucleotides is expressed, and
recovering said polypeptide. Within another embodiment the
polypeptide produced by the method is provided
[0016] Within another aspect the invention provides an isolated
polynucleotide comprising the polynucleotide sequence as shown in
SEQ ID NO:7.
[0017] Within another aspect the invention provides an antibody
that specifically binds to the isolated protein comprising residues
20 to 185 as shown in SEQ ID NO:8.
[0018] Within one aspect of the invention there is provided an
isolated polypeptide comprising fifteen contiguous amino acid
residues of a polypeptide as shown in SEQ ID NO:M, wherein M is
selected form the group consisting of 2, 5, and 8. Within one
embodiment, the at least fifteen contiguous amino acid residues of
SEQ ID NO:M are operably linked via a peptide bond or polypeptide
linker to a second polypeptide selected from the group consisting
of maltose binding protein, an immunoglobulin constant region, a
polyhistidine tag, and a peptide as shown in SEQ ID NO:10. Within
another embodiment, the polypeptide comprises at least 30
contiguous residues of SEQ ID NO:M. Within a further embodiment,
the polypeptide comprises at least 47 contiguous residues of SEQ ID
NO:M.
[0019] Within another aspect of the invention there are provided
polynucleotides encoding the polypeptides disclosed above. Within
certain embodiments of the invention the polynucleotides comprise a
sequence of nucleotides selected from the group consisting of 1, 4,
and 7.
[0020] The invention also provides an isolated polynucleotide
encoding a fusion protein, wherein the fusion protein comprises a
secretory peptide selected from the group consisting of residues 1
to 22 of SEQ ID NO:2; residues 1 to 18 of SEQ ID NO:5; and residues
1 to 19 of SEQ ID NO:8, and wherein the secretory peptide is
operably linked to a second polypeptide.
[0021] Within an additional aspect the invention provides a method
of detecting protein secretion from a cell or tissue comprising
detecting a mature MSP selected from the group consisting of SEQ ID
NO:M, wherein M is selected from the group consisting of 2, 5, and
8.
[0022] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0024] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification 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 82:7952-7954, 1985; see SEQ ID NO: 123), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-1210, 1988),
maltose binding protein (Kellerman and Ferenci, Methods Enzymol.
90:459-463, 1982; Guan et al., Gene 67:21-30, 1987), streptavidin
binding peptide, thioredoxin, ubiquitin, cellulose binding protein,
T7 polymerase, immunoglobulin constant domain, or other antigenic
epitope or binding domain. See, in general, Ford et al., Protein
Expression and Purification 2: 95-107, 1991. Affinity tags can be
used individually or in combination. DNAs encoding affinity tags
and other reagents are available from commercial suppliers (e.g.,
Pharmacia Biotech, Piscataway, N.J.; Eastman Kodak, New Haven,
Conn.; New England Biolabs, Beverly, Mass.).
[0025] 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.
[0026] 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.
[0027] A "complement" of a polynucleotide molecule is a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to
5'CCCGTGCAT 3'.
[0028] "Corresponding to", when used in reference to a nucleotide
or amino acid sequence, indicates the position in a second sequence
that aligns with the reference position when two sequences are
optimally aligned.
[0029] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons encompass different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0030] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription, wherein said segments are arranged
in a way that does not exist naturally. Such additional segments
include promoter and terminator sequences, and may also include one
or more origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0031] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0032] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide or protein is
substantially free of other polypeptides or proteins, particularly
other polypeptides or proteins of animal origin. It is preferred to
provide the polypeptides or proteins in a highly purified form,
i.e. greater than 95% pure, more preferably greater than 99% pure.
When used in this context, the term "Isolated" does not exclude the
presence of the same polypeptide or protein in alternative physical
forms, such as dimers or alternatively glycosylated or derivatized
forms.
[0033] A "mature protein" is a protein that is produced by cellular
processing of a primary translation product of a DNA sequence. Such
processing may include removal of a secretory signal peptide,
sometimes in combination with a propeptide. Mature sequences can be
predicted from full-length sequences using methods known in the art
for predicting cleavage sites. See, for example, von Heijne (Nuc.
Acids Res. 14:4683, 1986). The sequence of a mature protein can be
determined experimentally by expressing a DNA sequence of interest
in a eukaryotic host cell and determining the amino acid sequence
of the final product. For proteins lacking secretory peptides, the
primary translation product will be the mature protein.
[0034] "Operably linked", when referring to DNA segments, indicates
that the segments are arranged so that they function in concert for
their intended purposes, e.g., transcription initiates in the
promoter and proceeds through the coding segment to the terminator.
When referring to polypeptides, "operably linked" includes both
covalently (e.g., by disulfide bonding) and non-covalently (e.g.,
by hydrogen bonding, hydrophobic interactions, or salt-bridge
interactions) linked sequences; wherein the desired function(s) of
the sequences are retained.
[0035] 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.
[0036] "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.
[0037] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired. Such unpaired ends will
in general not exceed 20 nt in length.
[0038] 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".
[0039] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0040] 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.
[0041] A "secretory signal sequence" is a DNA sequence that encodes
a polypeptide (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.
[0042] All references cited herein are incorporated by reference in
their entirety.
[0043] The present invention is based in part upon the discovery of
a group of novel, protein-enoding DNA molecules, designated "MSP"
proteins, polypeptides and polynucleotides. Included in this group
are secreted proteins herein designated zcyto27, zsig96, and
ztth1.
[0044] A secretory peptide of a protein of the present invention
can be used to direct the secretion of other proteins of interest
from a host cell. Thus, the present invention provides, inter alia,
fusions comprising such a secretory peptide of a protein disclosed
herein operably linked to another protein of interest. The
secretory peptide can be used to direct the secretion of other
proteins of interest by joining a polynucleotide sequence encoding
it, in the correct reading frame, to the 5' end of a sequence
encoding the other protein of interest. Those skilled in the art
will recognize that the resulting fused sequence may encode
additional residues of a protein of the present invention at the
amino terminus of the protein to be secreted. In the extreme case,
the fusion may comprise an entire protein of the present invention
fused to the amino terminus of a second protein, whereby secretion
of the fusion protein is directed by the secretory peptide of the
protein of the present invention. It will often be desirable to
include a proteolytic cleavage site between the protein of the
present invention (or portion thereof) and the other protein of
interest. The joined polynucleotide sequences are then introduced
into a host cell, which is cultured according to conventional
methods. The protein of interest is then recovered from the culture
media. Methods for introducing DNA into host cells, culturing the
cells, and isolating recombinant proteins are known in the art.
Representative methods are summarized below.
[0045] Higher order structures of the proteins of the present
invention can be predicted by computer analysis using available
software (e.g., the Insight II.RTM. viewer and homology modeling
tools available from MSI, San Diego, Calif.; and King and
Sternberg, Protein Sci. 5:2298-310, 1996). In addition, analytical
algorithms permit the identification of homologies between newly
discovered proteins and known proteins. Such homologies are
indicative of related biological functions.
[0046] Zcyto27 is a member of a cytokine protein family and is
further shown herein in SEQ ID NO:1, polynucleotides; SEQ ID NO:2,
polypeptides; and SEQ ID NO:3, degenerated polynucleotide sequence.
Cytokines generally stimulate proliferation or differentiation of
cells of the hematopoietic lineage or participate in the immune and
inflammatory response mechanisms of the body. Examples of
cytokines, which affect hematopoiesis, are erythropoietin (EPO),
which stimulates the development of red blood cells; thrombopoietin
(TPO), which stimulates development of cells of the megakaryocyte
lineage; and granulocyte-colony stimulating factor (G-CSF), which
stimulates development of neutrophils. These cytokines are useful
in restoring normal blood cell levels in patients suffering from
anemia, thrombocytopenia, and neutropenia or receiving chemotherapy
for cancer.
[0047] In particular, Zcyto27 bears significant identity to
interleukin 2, interleukin 3, and interleukin 5, at 12, 13, and 12%
respectively. The interleukins are a family of cytokines that
mediate immunological responses, including inflammation. The
interleukins mediate a variety of inflammatory pathologies. Central
to an immune response is the T cell, which produce many cytokines
and adaptive immunity to antigens. Cytokines produced by the T cell
have been classified as type 1 and type 2 (Kelso, A. Immun. Cell
Biol. 76:300-317, 1998). Type 1 cytokines include IL-2,
IFN-.gamma., LT-.alpha., and are involved in inflammatory
responses, viral immunity, intracellular parasite immunity and
allograft rejection. Type 2 cytokines include IL-4, IL-5, IL-6,
IL-10 and IL-13, and are involved in humoral responses, helminth
immunity and allergic response. Shared cytokines between Type 1 and
2 include IL-3, GM-CSF and TNF-.alpha.. There is some evidence to
suggest that Type 1 and Type 2 producing T cell populations
preferentially migrate into different types of inflamed tissue.
[0048] As a protein that is expressed in kidney, liver and fetal
liver, PBL, prostate, spleen, testis, thymus, lymph node and bone
marrow, the polypeptides and polypeptides of Zcyto27 will find use
in modulating the immune reaction and inflammation in these and
other organs.
[0049] As a cytokine related to interleukin 3 and interleukin 5,
Zcyto27 has regions of the polypeptide sequence that will fold in
three-dimensional space to form helical regions. The boundaries of
these helices are based on alignment with il5 and il3, and one
skilled in the art would realize that such structural boundaries
should be +/-3 residues. The first helix (A) is from amino acid
position 22 to amino acid position 35 of SEQ ID NO:2. The second
(B), third (C), and fourth (D) helices are from amino acid 60 to
amino acid 73, from amino acid 84 to amino acid 95, and from amino
acid 113 to amino acid 130 as shown in SEQ ID NO:2,
respectively.
[0050] In designing and using antibodies to Zcyto27 to modulate its
action in inflammation and immune response, antibodies directed to
the helical regions of Zcyto27 as shown above, particularly,
antibodies against the first (A) and last (D) helices are
suggested.
[0051] Mature T cells may be activated, i.e., by antigen or other
stimulus, to produce, for example, cytokines, biochemical signaling
molecules, or receptors that further influence the fate of the T
cell population.
[0052] B cells can be activated via receptors on their cell surface
including B cell receptor and other accessory molecules to perform
accessory cell functions, such as production of cytokines.
[0053] The gene for Zcyto27 has been mapped to 14q24. As such,
polynucleotides encoding the polypeptide sequence of SEQ ID NO:2,
and complements thereof, may be useful to identify and further
study this location on the chromosome.
[0054] Also included in the proteins of the present invention, is a
secreted protein, Zsig96. Zsig96 is taught herein in SEQ ID NO:4,
polynucleotides; SEQ ID NO:5, polypeptides; and SEQ ID NO:6,
degenerated polynucleotide sequence.
[0055] Zsig96 is a secreted protein with specific tissue expression
in brain and pancreas tissues. As a potential Type II membrane
protein, a membrane bound form of Zsig96 can be used as a marker
for brain and pancreas tissues. Zsig96 is specifically not
expressed in a number of other tissues, including kidney, lung, and
skeletal muscle, among others.
[0056] As a small protein expressed in the brain and pancreas,
Zsig96 may be involved in nutritional homeostasis. Many such
regulatory peptides are found in the gastrointestinal environment.
These peptides may be synthesized in the digestive system and act
locally, but can also be active in the brain as well. In addition,
the reverse is also found, i.e., peptides are synthesized in the
brain, but found to regulate cells in the gastrointestinal tract.
This phenomena has been called the "brain-gut axis" and is
important for signaling satiety, regulating body temperature and
other physiological processes that require feedback between the
brain and gut. Other peptides which are involved in the brain-gut
axis may be expressed in other endocrine and exocrine tissues such
as the pancreas and pituitary.
[0057] Gut peptide hormones including gastrin, cholecystokinin
(CCK), secretin, gastric inhibitory peptide (GIP), vasoactive
intestinal polypeptide (VIP), motilin, somatostatin, pancreatic
peptide (PP), substance P and neuropeptide Y (NPY), use several
different mechanisms of action. For example, gastrin, motilin and
CCK function as endocrine- and neurocrine-type hormones. Others,
such as gastrin and GIP, are thought to act exclusively in an
endocrine fashion. Other modes of action include a combination of
endocrine, neurocrine and paracrine action (e.g., somatostatin);
exclusively neurocrine action (e.g., NPY); and a combination of
neurocrine and paracrine actions (e.g., VIP and Substance P). Most
of the gut hormone actions are mediated by membrane-bound receptors
and activate second messenger systems. For a review of gut peptides
see, Mulvihill et al., in Basic and Clinical Endocrinology,
pp.551-570, 4th edition Greenspan F. S. and Baxter, J. D. eds.,
Appleton & Lange, Norwalk, Conn., 1994.
[0058] Molecules of Zsig96 will be useful in modulation of nutrient
uptake, modulation of cell growth, proliferation and
differentiation, modulation of hormones, modulation of the
secretion of digestive enzymes, and/or modulation of secretion of
enzymes and/or hormones in the pancreas, and can be used to
modulate ligand-receptor binding or to treat or prevent development
of pathological conditions in such diverse tissue as stomach,
brain, pituitary, hypothalamus, hippocampus, duodenum, jejunum,
small intestine, and pancreas. In particular, certain diseases may
be amenable to such diagnosis, treatment or prevention. The
molecules of the present invention can be used to modulate
inhibition and proliferation of neurons and myocytes in stomach,
pituitary, hypothalamus, hippocampus, duodenum, jejunum, small
intestine, and pancreas. The polypeptides, nucleic acid and/or
antibodies of the present invention can be used in diagnosis,
treatment or prevention of disorders associated with, for example,
gastric reflux, gastroparesis, modulation of secretion of pituitary
hormones, including growth hormone, and or growth hormone
stimulating hormone, Crohn's Disease, metabolic wasting, gastric
ulcers, weight management, fertility, diseases of the developing
reproductive system, pacreatitis, diabetes, and degenerative
diseases.
[0059] The polypeptides, nucleic acid, and/or antibodies of Zsig96
may be used in treatment of disorders associated with pancreas,
diabetes, hypoglycemia; and
[0060] digestive systems including pancreas. The molecules of the
present invention may used to modulate or to treat or prevent
development of pathological conditions in such diverse tissue as
pancreas, and brain. In particular, certain syndromes or diseases
may be amenable to such diagnosis, treatment or prevention. Thus,
Zsig96 polypeptide pharmaceutical compositions of the present
invention may be useful in prevention or treatment of pancreatic
disorders associated with pathological regulation of the expansion
of neuroendocrine and exocrine cells in the pancreas, such as IDDM,
pancreatic cancer, pathological regulation of blood glucose levels,
insulin expression, insulin resistance or digestive function. The
Zsig96 polypeptides of the present invention may act in the
neuroendocrine/exocrine cell fate decision pathway and may
therefore be capable of regulating the expansion of neuroendocrine
and exocrine cells in the pancreas. One such regulatory use is that
of islet cell regeneration. Also, it has been hypothesized that the
autoimmunity that triggers IDDM starts in utero, and Zsig96
polypeptide is a developmental gene involved in cell partitioning.
Assays and animal models are known in the art for monitoring the
exocrine/neuroendocrine cell lineage decision, for observing
pancreatic cell balance and for evaluating Zsig96 polypeptide,
fragment, fusion protein, antibody, agonist or antagonist in the
prevention or treatment of the conditions set forth above.
[0061] The gene for Zsig96 has been mapped to 11p12. As such,
polynucleotides encoding the polypeptide sequence of SEQ ID NO:2,
and complements thereof, may be useful to identify and further
study this location on the chromosome.
[0062] Also included in the present invention in is a growth
factor, Ztth1, which is taught herein in SEQ ID NO:7,
polynucleotides; SEQ ID NO:8, polypeptides; and SEQ ID NO:9,
degenerated polynucleotide sequence.
[0063] Growth factors are important in the control of cellular
differentiation of multicellular organisms. These diffusable
molecules allow cells to communicate with each other and act in
concert to form tissues and organs, and to repair and regenerate
damaged tissue. Examples of growth factors include the interferons,
the interleukins, platelet derived growth factor, epidermal growth
factor, and granulocyte-macrophage colony stimulating factor, among
others.
[0064] Growth factors influence cellular metabolism by binding to
receptor proteins. Certain receptors are integral membrane proteins
that bind with the hormone or growth factor outside the cell, and
that are linked to signaling pathways within the, cell, such as
second messenger systems. Other classes of receptors are soluble
intracellular molecules.
[0065] Further as a secreted proteins, the polynucleotides of the
secretory signal sequences of Zcyto27, Zsig96, and Ztth1 can be
fused to a different protein of interest to direct the protein
through the secretory pathway of the cell. These secretory signal
sequences are from residue 1 to residue 22 of SEQ ID NO:2 for
Zcyto27; from residue 1 to residue 18 of SEQ ID NO:5 for Zsig96;
and from residue 1 to residue 19 of SEQ ID NO:8 for Ztth1.
[0066] A protein of the present invention can be prepared as a
fusion protein by joining it to a second polypeptide or a plurality
of additional polypeptides. Suitable second polypeptides include
amino- or carboxyl-terminal extensions, such as linker peptides of
up to about 20-25 residues and extensions that facilitate
purification (affinity tags) as disclosed above. A protein of
interest can be prepared as a fusion to a dimerizing protein as
disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred
dimerizing proteins in this regard include immunoglobulin constant
region domains. Immunoglobulin-polypeptide fusions can be expressed
in genetically engineered cells to produce a variety of multimeric
analogs of a protein of interest. Fusion proteins can also comprise
auxiliary domains that target the protein of interest to specific
cells, tissues, or macromolecules (e.g., collagen). For example, a
protein of interest can be targeted to a predetermined cell type by
fusing it to a ligand that specifically binds to a receptor on the
surface of a target cell. In this way, proteins can be targeted for
therapeutic or diagnostic purposes. A protein can be fused to two
or more moieties, such as an affinity tag for purification and a
targeting domain. Protein fusions can also comprise one or more
cleavage sites, particularly between domains. See, Tuan et al.,
Connective Tissue Research 34:1-9, 1996. Proteins of the present
invention can also be used as targetting moieties within fusion
proteins comprising, for example, cytokines, cytotoxins, or other
biologically active polypeptide moieties.
[0067] Protein fusions of the present invention will usually
contain not more than about 1,200 amino acid residues joined to the
MSP protein. For example, an MSP protein can be fused to E. coli
.beta.-galactosidase (1,021 residues; see Casadaban et al., J.
Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue
factor Xa cleavage site. Such a protein comprising, for example,
MSPZ809335G4P (SEQ ID NO:100), contains 723 amino acid residues. In
a second example, an MSP protein can be fused to maltose binding
protein (approximately 370 residues), a 4-residue cleavage site,
and a 6-residue polyhistidine tag.
[0068] As disclosed above, the proteins of the present invention or
portions thereof can also be used to direct the secretion of a
second protein. When such fusions are designed so that the secreted
protein retains a portion of the protein of the present invention,
the fusion protein can be purified by means that exploit the
properties of the protein of the present invention. Typical of such
methods is immunoaffinity chromatography using an antibody directed
against a protein of the present invention. When such a fusion is
engineered to contain a cleavage site at the fusion point, the
fusion can be cleaved and the protein of interest recovered free of
extraneous sequence.
[0069] As secreted proteins, MSP can be useful to monitor secretion
of proteins in general from cells or tissue. In doing so, cell
lines or tissues shown to express the proteins of the present
invention can be monitored to detect for example 1f the cell or
tissue has a defective secretory pathway. Thus, cell membrane
extracts or conditioned media from the cell or tissue can be tested
for the presence or absence of the mature MSP protein. Similarly,
when trying to identify if a recombinantly expressed protein is has
a functional signal peptide, the molecules of the present invention
can be used as a positive control.
[0070] The present invention also provides polynucleotide
molecules, including DNA and RNA molecules, that encode the
proteins disclosed above. 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. The amino acid sequence information
provided herein can be used by one of ordinary skill in the art to
generate degenerate sequences comprising all nucleotide sequences
encoding a particular polypeptide. Table 1 sets forth the
one-letter codes used 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 Resolutions Complement Resolutions 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.vertlin- e.T N
A.vertline.C.vertline.G.vertline.T
[0071] Degenerate codons encompassing all possible codons for a
given amino acid are set forth in Table 2, below.
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
CAN 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 Gap -
---
[0072] 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 each 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 sequences 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 sequences disclosed in the
accompanying Sequence Listing.
[0073] Methods for preparing DNA and RNA are well known in the art.
Complementary DNA (cDNA) clones are prepared from RNA that is
isolated from a tissue or cell that produces large amounts of the
cognate mRNA. Such tissues and cells are identified by methods
commonly known in the art, such as Northern blotting (Thomas, Proc.
Natl. Acad. Sci. USA 77:5201, 1980). Databases of expressed
sequence tags (ESTs) can be analyzed to produce an "electronic
Northern" wherein sequences are assigned to specific cell or tissue
sources on the basis of their abundance within libraries.
[0074] A panel of cDNAs from human tissues can be screened for
tissue expression using PCR. As an example, such a panel can made
from commercially available first strand cDNA (Clontech
laboratories, Inc., Palo Alto Calif.) and would contain 20
first-strand cDNA samples from the human tissues shown in Table 6.
The panel can be set up in a 96-well format that further included a
human genomic DNA (obtained from Clontech Laboratories, Inc.)
positive control sample and a water-only well as a negative control
sample. Each well can contain approximately 0.2-100 pg/.mu.l of
cDNA, diluted with water to 17.5 .mu.l. The PCR reactions are set
up by adding oligonucleotide primers, DNA polymerase (Ex Taq.TM.;
TAKARA Shuzo Co. Ltd. Biomedicals Group, Japan or Advantage.TM. 2
cDNA polymerase mix; Clontech Laboratories, Inc.) with the
appropriate supplied buffer, dNTP mix (TAKARA Shuzo Co. Ltd.), and
a density increasing agent and tracking dye (RediLoad; Research
Genetics, Inc., Huntsville, Ala.) to each sample on the panel. The
amplification of the cDNA can be carried out as follows: incubation
at 94.degree. C. for 2 minutes; 35 cycles of 94.degree. C. for 30
seconds, 60.degree. C. for 20 seconds, and 72.degree. C. for 30
seconds; followed by incubation at 72.degree. C. for 5 minutes. A
portion of the PCR reaction product is then subjected to standard
agarose gel electrophoresis using a 4% agarose gel.
[0075] Total RNA can be prepared using guanidine HCl extraction
followed by isolation by centrifugation in a CsCl gradient
(Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A).sup.+ RNA
is prepared from total RNA using the method of Aviv and Leder
(Proc. Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA
(cDNA) is prepared from poly(A).sup.+ RNA using known methods. In
the alternative, genomic DNA can be isolated. For some applications
(e.g., expression in transgenic animals) it may be preferable to
use a genomic clone, or to modify a cDNA clone to include at least
one genomic intron. Methods for identifying and isolating cDNA and
genomic clones are well known and within the level of ordinary
skill in the art, and include the use of the sequences disclosed
herein, sequences complementary thereto, or parts thereof, for
probing or priming a library. Such methods include, for example,
hybridization or polymerase chain reaction ("PCR", Mullis, U.S.
Pat. No. 4,683,202). Expression libraries can be probed with
antibodies to a protein of interest, receptor fragments, or other
specific binding partners.
[0076] The polynucleotides of the present invention can also be
prepared by automated synthesis. Synthesis of polynucleotides is
within the level of ordinary skill in the art, and suitable
equipment and reagents are available from commercial suppliers.
See, in general, Glick and Pasternak, Molecular Biotechnology,
Principles & Applications of Recombinant DNA, ASM Press,
Washington, D.C., 1994; Itakura et al., Ann. Rev. Biochem. 53:
323-56, 1984; and Climie et al., Proc. Natl. Acad. Sci. USA
87:633-7, 1990.
[0077] The present invention further provides antisense
polynucleotides that are complementary to a segment of a
polynucleotide as set forth in one of SEQ ID NO:N, wherein N is an
odd integer from 1 to 327. Such antisense polynucleotides are
designed to bind to the corresponding mRNA and inhibit its
translation. Antisense polynucleotides are used to inhibit gene
expression in cell culture or in a patient, and can be used as
probes or primers for research or diagnostic purposes.
[0078] Probes and primers of the present invention comprise a
suitable fragment, and may comprise up to the complete sequence, of
a polynucleotide as shown in SEQ ID NO:N or the complement thereof,
wherein N is an odd integer from 1 to 327. Probes will generally be
at least 20 nucleotides in length, although somewhat shorter probes
(14-17 nucleotides)-can be used. PCR primers are at least 5
nucleotides in length, preferably 15 or more nt, more preferably
20-30 nt. Shorter polynucleotide probes and primers are referred to
in the art as "oligonucleotides," and can be DNA or RNA. Probes
will generally comprise an oligonucleotide linked to a label, such
as a radionuclide.
[0079] Probes and primers as disclosed herein can be used for
cloning allelic, orthologous, and paralogous sequences. Allelic
variants of the disclosed sequences can be cloned by probing cDNA
or genomic libraries from different individuals according to
standard procedures. Orthologous sequences 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 the protein. 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 cDNA can then be isolated by a
variety of methods, such as by probing with a complete or partial
human cDNA or with one or more sets of degenerate probes based on
the disclosed sequences. A cDNA can also be cloned by PCR using
primers designed from the 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 the encoded protein. Similar
techniques can also be applied to the isolation of genomic clones.
Orthologous and paralogous sequences can be identified from
libraries by probing blots at low stringency and washing the blots
at successively higher stringency until background is suitably
reduced.
[0080] Probes and primers disclosed herein can be used to clone 5'
non-coding regions of a corresponding gene. Such promoter elements
can thus be used to direct the tissue-specific expression of
heterologous genes in, for example, transgenic animals or patients
treated with gene therapy. Cloning of 5' flanking sequences also
facilitates production of a protein of interest by "gene
activation" as disclosed in U.S. Pat. No. 5,641,670. Briefly,
expression of an endogenous gene in a cell is altered by
introducing into its locus a DNA construct comprising at least a
targeting sequences a regulatory sequence, an exon, and an unpaired
splice donor site. The targeting sequence is a 5' non-coding
sequence that permits homologous recombination of the construct
with the endogenous locus, whereby the sequences within the
construct become operably linked with the endogenous coding
sequence. In this way, an endogenous promoter can be replaced or
supplemented with other regulatory sequences to provide enhanced,
tissue-specific, or otherwise regulated expression.
[0081] The polynucleotides of the present invention further include
polynucleotides encoding the fusion proteins, including signal
peptide fusions, disclosed above.
[0082] In studying cell biology it is useful to monitor changing
levels of mRNA as a function of cell and tissue development. As a
set of cDNA molecules, the polynucleotides of the present
invention, for example SEQ ID NO:N, wherein N is an odd integer
from 1 to 327, can also be used to screen for levels of mRNA of the
polypeptides, i.e., as shown in SEQ ID NO:M, wherein M is selected
form the group consisting of 2, 5, and 8. Screening for message
level fluctuations of the molecules of the present invention are
useful for studying, for example, cell replication, activation, and
quiescence.
[0083] The present invention further provides a computer-readable
medium encoded with a data structure that provides at least one of
SEQ ID NO:1 through SEQ ID NO:9. 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.RTM. 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).
[0084] The polypeptides of the present invention, including
full-length proteins, biologically active fragments, immunogenic
fragments, and fusion proteins, can be produced in genetically
engineered host cells according to conventional techniques.
Suitable host cells are those cell types that can be transformed or
transfected with exogenous DNA and grown in culture, and include
bacteria, fungal cells, and cultured higher eukaryotic cells.
Eukaryotic cells, particularly cultured cells of multicellular
organisms, are generally preferred for the production of proteins
having higher eukaryotic-type post-translational modifications
(e.g., .gamma.-carboxylation) and for making proteins, especially
secretory proteins, for pharmaceutical use in humans. Techniques
for manipulating cloned DNA molecules and introducing exogenous DNA
into a variety of host cells are disclosed by Sambrook et al.,
Molecular. Cloning: A Laboratory Manual, 2nd ed., Cold Spring
Harbor Laboratory. Press, Cold Spring Harbor, N.Y., 1989, and
Ausubel et al., eds., Current Protocols in Molecular Biology, Green
and Wiley and Sons, NY, 1993.
[0085] In general, a DNA sequence encoding a polypeptide of
interest is operably linked to other genetic elements required for
its expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers can be
provided on separate vectors, and replication of the exogenous DNA
can be achieved through integration into the host cell genome.
Selection of promoters, terminators, selectable markers, vectors
and other elements is a matter of routine design within the level
of ordinary skill in the art. Many such elements are described in
the literature and are available through commercial suppliers.
[0086] To direct a polypeptide into the secretory pathway of a host
cell, a secretory signal sequence (also known as a leader sequence,
prepro sequence or pre sequence) is provided in the expression
vector. The secretory signal sequence may be that of the protein of
interest, or may be derived from another secreted protein (e.g.,
t-PA; see U.S. Pat. No. 5,641,655) or synthesized de novo. The
secretory signal sequence is operably linked to the DNA sequence
encoding the protein of interest, i.e., the two sequences are
joined in the correct reading frame and positioned to direct the
newly synthesized protein into the secretory pathway of the host
cell. Secretory signal sequences are commonly positioned 5' to the
DNA sequence encoding the protein of interest, although certain
secretory signal sequences may be positioned elsewhere in the DNA
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).
[0087] Cultured mammalian cells are suitable hosts for use within
the present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., ibid.), and liposome-mediated transfection (Hawley-Nelson et
al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The
production of recombinant polypeptides in cultured mammalian cells
is disclosed by, for example, Levinson et al., U.S. Pat. No.
4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al.,
U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134.
Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL
1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632),BHK 570
(ATCC No. CRL 10314), 293 (ATCC. No. CRL 1573; Graham et al., J.
Gen. Viriol. 36:59-72, 1977) and Chinese hamster ovary (e.g.
CH0-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines
are known in the art and available, from public depositories such
as the American Type Culture Collection, Manasas, Va. In general,
strong transcription promoters are preferred, such as promoters
from SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288.
Other suitable promoters include those from metallothionein genes
(U.S. Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major
late promoter. Within an alternative embodiment, adenovirus vectors
can be employed. See, for example, Gamier et al., Cytotechnol.
15:145-55, 1994.
[0088] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." An exemplary selectable marker is a gene
encoding resistance to the antibiotic neomycin. 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.
[0089] Insect cells can be infected with recombinant baculovirus,
commonly derived from Autographa californica nuclear polyhedrosis
virus (AcNPV). See, King and Possee, The Baculovirus Expression
System: A Laboratory Guide, London, Chapman & Hall; O'Reilly et
al., Baculovirus Expression Vectors: A Laboratory Manual, New York,
Oxford University Press., 1994; and Richardson, Ed., Baculovirus
Expression Protocols. Methods in Molecular Biology, Humana Press,
Totowa, N.J., 1995. Recombinant baculovirus can also be produced
through the use of a transposon-based system described by Luckow et
al. (J. Virol. 67:4566-4579, 1993). This system, which utilizes
transfer vectors, is commercially available in kit form
(Bac-to-Bac.TM. kit; Life Technologies, Rockville, Md.). See also,
Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990; Bonning
et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk and
Rapoport, J. Biol. Chem. 270:1543-1549, 1995.
[0090] For protein production, the recombinant virus is used to
infect host cells, typically, a cell line derived from the fall
armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or
Trichoplusia ni (e.g., High Five.TM. cells; Invitrogen, Carlsbad,
Calif.). See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. See also, U.S. Pat. No. 5,300,435.
Serum-free media are used to grow and maintain the cells. Suitable
media formulations are known in the art and can be obtained from
commercial suppliers. The cells are 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. Procedures used are generally described in
available laboratory manuals (e.g., King and Possee, ibid.;
O'Reilly et al., ibid.; Richardson, ibid.). See also, Guarino et
al., U.S. Pat. No. 5,162,222 and WIPO publication WO 94/06463.
[0091] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. 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). A preferred 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.
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.
[0092] 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.
132:3459-3465, 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. Production of recombinant,
proteins in Pichia methanolica is disclosed in U.S. Pat. Nos.
5,716,808, 5,736,383, 5,854,039, and 5,888,768; and WIPO
publications WO 99/14347 and WO 99/14320.
[0093] Other higher eukaryotic cells, including plant cells and
avian cells, can also be used as hosts according to methods
commonly known in the art. For example, the use of Agrobacterium
rhizogenes as a vector for expressing genes in plant cells has been
reviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58,
1987.
[0094] Prokaryotic host cells, including strains of the bacteria
Escherichia coli, Bacillus and other genera are also useful host
cells within the present invention. Techniques for transforming
these hosts and expressing foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a 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.
[0095] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell.
[0096] It is preferred to purify the polypeptides and proteins of
the present invention to .gtoreq.80% purity, more preferably to
.gtoreq.90% purity, even more preferably .gtoreq.95% purity, and
particularly preferred is a pharmaceutically pure state, that is
greater than 99.9% pure with respect to contaminating
macromolecules, particularly other proteins and nucleic acids, and
free of infectious and pyrogenic agents. Preferably, a purified
polypeptide or protein is substantially free of other polypeptides
or proteins, particularly those of animal origin.
[0097] Expressed recombinant proteins (including single polypeptide
chains, chimeric polypeptides., and polypeptide multimers) are
purified by conventional protein purification methods, typically by
a combination of chromatographic techniques. See in general,
Affinity Chromatography: Principles & Methods, Pharmacia LKB
Biotechnology, Uppsala, Sweden, 1988; and Scopes, Protein
Purification: Principles and Practice, Springer-Verlag, New York,
1994. Proteins comprising a polyhistidine affinity tag (typically
about 6 histidine residues) are purified by affinity chromatography
on a nickel chelate resin. See, for example, Houchuli et al.,
Bio/Technol. 6: 1321-1325, 1988. Proteins comprising a glu-glu tag
can be purified by immunoaffinity chromatography essentially as
disclosed by Grussenmeyer et al., ibid. Proteins comprising other
affinity tags can be purified by appropriate affinity
chromatography methods, which are known in the art.
[0098] Proteins of the present invention and fragments thereof can
also be prepared through chemical synthesis according to methods
known in the art, including exclusive solid phase synthesis,
partial solid phase methods, fragment condensation or classical
solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc.
85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2nd
edition), Pierce Chemical Co., Rockford, Ill., 1984; Bayer and
Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase
Peptide Synthesis: A Practical Approach, IRL Press, Oxford,
1989.
[0099] Using methods known in the art, the proteins of the present
invention can be prepared in a variety of modified or derivatized
forms. For example, the proteins can be prepared glycosylated or
non-glycosylated; pegylated or non-pegylated; and may or may not
include an initial methionine amino acid residue.
[0100] Biological activities of the proteins of the present
invention can be measured in vitro using cultured cells or in vivo
by administering molecules of the claimed invention to the
appropriate animal model. Many such assays and models are known in
the art. Guidance in initial assay selection is provided by
structural predictions and sequence alignments. However, even if no
functional prediction is made, the activity of a protein can be
elucidated by known methods, including, for example, screening a
variety of target cells for a biological response, other in vitro
assays, expression in a host animal, or through the use of
transgenic and/or "knockout" animals. Through the application of
robotics, many in vitro assays can be adapted to rapid,
high-throughput screeing of a large number of samples. Target cells
for use in activity assays include, without limitation, vascular
cells (especially endothelial cells and smooth muscle cells),
hematopoietic., (myeloid and lymphoid) cells, liver cells
(including hepatocytes, fenestrated endothelial cells, Kupffer
cells, and Ito cells), fibroblasts (including human dermal
fibroblasts and lung fibroblasts), neurite cells (including
astrocytes, glial cells, dendritic cells, and PC-12 cells), fetal
lung cells, articular synoviocytes, pericytes, chondrocytes,
osteoblasts, adipocytes, and prostate epithelial cells. Endothelial
cells and hematopoietic cells are derived from a common ancestral
cell, the hemangioblast (Choi et al., Development 125:725-732,
1998).
[0101] Biological activity can be measured with a silicon-based
biosensor microphysiometer that measures the extracellular
acidification rate or proton excretion associated with receptor
binding and subsequent physiologic cellular responses. An exemplary
such device is the Cytosensor.TM. Microphysiometer manufactured by
Molecular Devices, Sunnyvale, Calif. A variety of cellular
responses, such as cell proliferation, ion transport, energy
production, inflammatory response, regulatory and receptor
activation, and the like, can be measured by this method. See, for
example, McConnell et al., Science 257:1906-1912, 1992; Pitchford
et al., Meth. Enzymol. 228:84-108, 1997; Arimilli et al., J.
Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.
Pharmacol. 346:87-95, 1998. The microphysiometer can be used for
assaying adherent or non-adherent eukaryotic or prokaryotic cells.
By measuring extracellular acidification changes in cell media over
time, the microphysiometer directly measures cellular responses to
various stimuli, including agonistic and antagonistic stimuli.
Preferably, the microphysiometer is used to measure responses of a
eukaryotic cell known to be responsive to the protein of interest,
compared to a control eukaryotic cell that does not respond to the
protein of interest. Responsive eukaryotic cells comprise cells
into which a receptor for the protein of interest has been
transfected, as well as naturally responsive cells. Differences in
the response of cells exposed to the protein of interest, relative
to a control not so exposed, are a direct measurement of
protein-modulated cellular responses. Such responses can be assayed
under a variety of stimuli. The present invention thus provides
methods of identifying agonists and antagonists of proteins of
interest, comprising providing cells responsive to a selected
protein, culturing a first portion of the cells in the absence of a
test compound, culturing a second portion of the cells in the
presence of a test compound, and detecting a change in a cellular
response of the second portion of the cells as compared to the
first portion of the cells. The change in cellular response is
shown as a measurable change in extracellular acidification rate.
Culturing a third portion of the cells in the presence of the
protein of interest and the absence of a test compound provides a
positive control and a control to compare the agonist activity of a
test compound with that of the protein of interest. Antagonists can
be identified by exposing the cells to the protein of interest in
the presence and absence of the test compound, whereby a reduction
in protein-stimulated activity is indicative of antagonist activity
in the test compound.
[0102] As expressed secreted proteins the MSP polypeptides
disclosed herein can be used to screen for cell metabolism
effecting receptors. Thus, the polypeptides of the present
invention are useful for identifying new target receptors and drug
design. A subset of the polypeptides disclosed herein can include
membrane-bound receptors. This subset can include amongst other
receptors, homologs of G protein coupled receptors, which are
important mediators for cell activities including the synthesis of
second messengers. Thus a set of these G protein couple receptors
can mediate cel differentiation and proliferation, or hormone
expression in vitro and in vivo.
[0103] 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 (as disclosed by, e.g.,
Raines and Ross, Methods Enzymol. 109:749-773, 1985; Wahl et al.,
Mol. Cell Biol. 8:5016-5025, 1988; and 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).
Differentiation can be assayed using suitable precursor cells that
can be induced to differentiate into a more mature phenotype.
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). Effects of a protein on tumor cell growth and
metastasis can be analyzed using the Lewis lung carcinoma model,
for example as described by Cao et al., J. Exp. Med. 182:2069-2077,
1995. Activity of a protein on cells of neural origin can be
analyzed using assays that measure effects on neurite growth as
disclosed below.
[0104] In vitro assays for pro- and anti-inflammatory activity are
known in the art. Exemplary activity assays include mitogenesis
assays in which IL-1 responsive cells (e.g., D10.N4.M cells) are
incubated in the presence of IL-1 or a test protein for 72 hours at
37.degree. C. in a 5% CO.sub.2 atmosphere. IL-2 (and optionally
IL-4) is added to the culture medium to enhance sensitivity and
specificity of the assay. .sup.3H-thymidine is then added, and
incubation is continued for six hours. The amount of label
incorporated is indicative of agonist activity. See, Hopkins and
Humphreys, J. Immunol. Methods 120:271-276, 1989; Greenfeder et
al., J. Biol. Chem. 270:22460-22466, 1995. Stimulation of cell
proliferation can also be measured using thymocytes cultured in a
test protein in combination with phytohemagglutinin. IL-1 is used
as a control. Proliferation is detected as .sup.3H-thymidine
incorporation or metabolic breakdown of (MTT) (Mosman, ibid.).
[0105] Protein activity may also be detected using assays designed
to measure induction of one or more growth factors- or other
macromclecules. Preferred such assays include those for determining
the presence of hepatocyte growth factor (HGF), epidermal growth
factor (EGF), transforming growth factor alpha (TGF.alpha.),
interleukin-6 (IL-6), VEGF, acidic fibroblast growth factor (aFGF),
angiogenin, and other macromolecules produced by the liver.
Suitable assays include mitogenesis assays using target cells
responsive to the macromolecule of interest, receptor-binding
assays, competition binding assays, immunological assays (e.g.,
ELISA), and other formats known in the art. Metalloprotease
secretion is measured from treated primary human dermal
fibroblasts, synoviocytes and chondrocytes. The relative levels of
collagenase, gelatinase and stromalysin produced in response to
culturing a target cell in the presence of a protein of interest is
measured using zymogram gels (Loita and Stetler-Stevenson, Cancer
Biology 1:96-106, 1990). Procollagen/collagen synthesis by dermal
fibroblasts and chondrocytes in response to a test protein is
measured using .sup.3H-proline incorporation into nascent secreted
collagen. .sup.3H-labeled collagen is visualized by SDS-PAGE
followed by autoradiography (Unemori and Amento, J. Biol. Chem.
265: 10681-10685, 1990). Glycosaminoglycan (GAG) secretion from
dermal fibroblasts and chondrocytes is measured using a
1,9-dimethylmethylene blue dye binding assay (Farndale et al.,
Biochim. Biophys. Acta 883:173-177, 1986). Collagen and GAG assays
are also carried out in the presence of IL-1.beta. or TGF-.beta. to
examine the ability of a protein to modify the established
responses to these cytokines.
[0106] Monocyte activation assays are carried out (1) to look for
the ability of a protein of interest to further stimulate monocyte
activation, and (2) to examine the ability of a protein of interest
to modulate attachment-induced or endotoxin-induced monocyte
activation (Fuhlbrigge et al., J. Immunol. 138: 3799-3802, 1987).
IL-1.beta. and TNF.alpha. levels produced in response to activation
are measured by ELISA (Biosource, Inc. Camarillo, Calif.).
Monocyte/macrophage cells, by virtue of CD14 (LPS receptor), are
exquisitely sensitive to endotoxin, and proteins with moderate
levels of endotoxin-like activity will activate these cells.
[0107] Other metabolic effects of proteins can be measured by
culturing target cells in the presence and absence of a protein and
observing changes in adipogenesis, gluconeogenesis, glycogenolysis,
lipogenesis, glucose uptake, or the like. Suitable assays are known
in the art.
[0108] Hematopoietic activity of proteins can be assayed on various
hematopoietic cells in culture. Preferred assays include primary
bone marrow colony assays and later stage lineage-restricted colony
assays, which are known in the art (e.g., Holly et al., WIPO
Publication WO 95/21920). Marrow cells plated on a suitable
semi-solid medium (e.g., 50% methylcellulose containing 15% fetal
bovine serum, 10% bovine serum albumin, and 0.6% PSN antibiotic
mix) are incubated in the presence of test polypeptide, then
examined microscopically for colony formation. Known hematopoietic
factors are used as controls. Mitogenic activity of a protein of
interest on hematopoietic cell lines can be measured as disclosed
above.
[0109] Cell migration is assayed essentially as disclosed by Khler
et al. (Arteriosclerosis, Thrombosis, and Vascular Biology
17:932-939, 1997). A protein is considered to be chemotactic if it
induces migration of cells from an area of low protein
concentration to an area of high protein concentration. A typical
assay is performed using modified Boyden chambers with a
polystryrene membrane separating the two chambers (Transwell;
Corning Costar Corp.). The test sample, diluted in medium
containing 1% BSA, is added to the lower chamber of a 24-well plate
containing Transwells. Cells are then placed on the Transwell
insert that has been pretreated with 0.2% gelatin. Cell migration
is measured after 4 hours of incubation at 37.degree. C.
Non-migrating cells are wiped off the top of the Transwell
membrane, and cells attached to the lower face of the membrane are
fixed and stained with 0.1% crystal violet. Stained cells are then
extracted with 10% acetic acid and absorbance is measured at 600
nm. Migration is then calculated from a standard calibration curve.
Cell migration can also be measured using the matrigel method of
Grant et al. ("Angiogenesis as a component of
epithelial-mesenchymal interactions" in Goldberg and Rosen,
Epithelial-Mesenchymal Interaction in Cancer, Birkhuser Verlag,
1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997).
[0110] Proteins can be assayed for the ability to modulate axon
guidance and growth. Suitable assays that detect changes in neuron
growth patterns include, for example, those disclosed in Hastings,
WIPO Publication WO 97/29189 and Walter et al., Development
101:685-96, 1987. Assays to measure the effects on neuron growth
are well known in the art. For example, the C assay (e.g., Raper
and Kapfhammer, Neuron 4:21-9, 1990 and Luo et al., Cell 75:217-27,
1993) can be used to determine collapsing activity of a protein of
interest on growing neurons. Other methods that can assess
protein-induced inhibition of neurite extension or divert such
extension are also known. See, Goodman, Annu. Rev. Neurosci.
19:341-77, 1996. Conditioned media from cells expressing a protein
of interest, or aggregates of such cells, can by placed in a gel
matrix near suitable neural cells, such as dorsal root ganglia
(DRG) or sympathetic ganglia explants, which have been co-cultured
with nerve growth factor. Compared to control cells,
protein-induced changes in neuron growth can be measured (as
disclosed by, for example, Messersmith et al., Neuron 14:949-59,
1995 and Puschel et al., Neuron 14:941-8, 1995). Neurite outgrowth
can be measured using neuronal cell suspensions grown in the
presence of molecules of the present invention. See, for example,
O'Shea et al., Neuron 7:231-7, 1991 and DeFreitas et al., Neuron
15:333-43, 1995.
[0111] Cell adhesion activity is assayed essentially as disclosed
by LaFleur et al. (J. Biol. Chem. 272:32798-32803, 1997). Briefly,
microtiter plates are coated with the test protein, non-specific
sites are blocked with BSA, and cells (such as smooth muscle cells,
leukocytes, or endothelial cells) are plated at a density of
approximately 10.sup.4-10.sup.5 cells/well. The wells are incubated
at 37.degree. C. (typically for about 60 minutes), then
non-adherent cells are removed by gentle washing. Adhered cells are
quantitated by conventional methods (e.g., by staining with crystal
violet, lysing the cells, and determining the optical density of
the lysate). Control wells are coated with a known adhesive
protein, such as fibronectin or vitronectin.
[0112] Assays for angiogenic activity are also known in the art.
For example, the effect of a protein of interest on primordial
endothelial cells in angiogenesis can be assayed in the chick
chorioallantoic membrane angiogenesis assay (Leung, Science
246:1306-1309, 1989; Ferrara, Ann. NY Acad. Sci. 752:246-256,
1995). Briefly, a small window is cut into the shell of an
eight-day old fertilized egg, and a test substance is applied to
the chorioallantoic membrane. After 72 hours, the membrane is
examined for neovascularization. Other suitable assays include
microinjection of early stage quail (Coturnix coturnix japonica)
embryos as disclosed by Drake et al. (Proc. Natl. Acad. Sci. USA
92:7657-7661, 1995); the rodent model of corneal neovascularization
disclosed by Muthukkaruppan and Auerbach (Science 205:1416-1418,
1979), wherein a test substance is inserted into a pocket in the
cornea of an inbred mouse; and the hampster cheek pouch assay
(Hockel et al., Arch. Surg. 128:423-429, 1993). Induction of
vascular permeability, which is indicative of angiogenic activity,
is measured in assays designed to detect leakage of protein from
the vasculature of a test animal (e.g., mouse or guinea pig) after
administration of a test compound (Miles and Miles, J. Physiol.
118:228-257, 1952; Feng et al., J. Exp. Med. 183:1981-1986, 1996).
In vitro assays for angiogenic activity include the tridimensional
collagen gel matrix model (Pepper et al. Biochem. Biophys. Res.
Comm. 189:824-831, 1992 and Ferrara et al., Ann. NY Acad. Sci.
732:246-256, 1995), which measures the formation of tube-like
structures by microvascular endothelial cells; and matrigel models
(Grant et al., "Angiogenesis as a component of
epithelial-mesenchymal interactions" in Goldberg and Rosen,
Epithelial-Mesenchymal Interaction in Cancer, Birkhuser Verlag,
1995, 235-248; Baatout, Anticancer Research 17:451-456, 1997),
which are used to determine effects on cell migration and tube
formation by endothelial cells seeded in matrigel, a basement
membrane extract enriched in laminin. It is preferred to carry out
angiogenesis assays in the presence and absence of vascular
endothelial growth factor (VEGF) to assess possible combinatorial
effects. It is also preferred to use VEGF as a control within in
vivo assays.
[0113] Receptor binding can be measured by the competition binding
method of Labriola-Tompkins et al., Proc. Natl. Acad. Sci. USA
88:11182-11186, 1991. In an exemplary assay for IL-1 receptor
binding, membranes pepared from EL-4 thymoma cells (Paganelli et
al., J. Immunol. 138:2249-2253, 1987) are incubated in the presence
of the test protein for 30 minutes at 37.degree. C. Labeled
IL-1.alpha. or IL-1.beta. is then added and the incubation is
continued for 60 minutes. The assay is terminated by membrane
filtration. The amount of bound label is determined by conventional
means (e.g., y counter). In an alternative assay, the ability of a
test protein to compete with labeled IL-1 for binding to cultured
human dermal fibroblasts is measured according to the method of
Dower et al. (Nature 324:266-268, 1986). Briefly, cells are
incubated in a round-bottomed, 96-well plate in a suitable culture
medium (e.g., RPMI 1640 containing 1% BSA, 0.1% Na azide, and 20 mM
HEPES pH 7.4) at 8.degree. C. on a rocker platform in the presence
of labeled IL-1. Various concentrations of test protein are added.
After the incubation (typically about two hours), cells are
separated from unbound label by centrifuging 60-.mu.l aliquots
through 200 .mu.l of phthalate oils in 400-.mu.l polyethylene
centrifuge tubes and excising the tips of the tubes with a razor
blade as disclosed by Segal and Hurwitz, J. Immunol. 118:1338-1347,
1977. Receptor binding assays for other cell types are known in the
art. See, for example, Bowen-Pope and Ross, Methods Enzymol.
109:69-100, 1985.
[0114] Receptor binding can also be measured using immobilized
receptors or ligand-binding receptor fragments. For example, an
immobilized receptor can be exposed to its labeled ligand and
unlabeled test protein, whereby a reduction in labeled ligand
binding compared to a control is indicative of receptor-binding
activity in the test protein. Within another format, a receptor or
ligand-binding receptor fragment is immobilized on a biosensor
(e.g., BIACore.TM., Pharmacia Biosensor, Piscataway, N.J.) and
binding is determined. Antagonists of the native ligand will
exhibit receptor binding but will exhibit essentially no activity
in appropriate activity assays or will reduce the ligand-mediated
response when combined with the native ligand. In view of the low
level of receptor occupancy required to produce a response to some
ligands. (e.g., IL-1), a large excess of antagonist (typically a
10- to 1000-fold molar excess) may be necessary to neutralize
ligand activity.
[0115] Receptor activation can be detected in target cells by: (1)
measurement of adenylate cyclase activity (Salomon et al., Anal.
Biochem. 58:541-48, 1974; Alvarez and Daniels, Anal. Biochem.
187:98-103, 1990); (2) measurement of change in intracellular cAMP
levels using conventional radioimmunoassay methods (Steiner et al.,
J. Biol. Chem. 247:1106-13, 1972; Harper and Brooker, J. Cyc. Nucl.
Res. 1:207-18, 1975); or (3) through use of a cAMP scintillation
proximity assay (SPA) method (such as available from Amersham
Corp., Arlington Heights, Ill.).
[0116] Proteins can be tested for serine protease activity or
proteinase inhibitory activity using conventional assays. Substrate
cleavage is conveniently assayed using a tetrapeptide that mimics
the cleavage site of the natural substrate and which is linked, via
a peptide bond, to a carboxyl-terminal para-nitro-anilide (pNA)
group. The protease hydrolyzes the bond between the fourth amino
acid residue and the pNA group, causing the pNA group to undergo a
dramatic increase in absorbance at 405 nm. Suitable substrates can
be synthesized according to known methods or obtained from
commercial suppliers. Inhibitory activity is measured by adding a
test sample to a reaction mixture containing enzyme and substrate,
and comparing the observed enzyme activity to a control (without
the test sample). A variety of such assays are known in the art,
including assays measuring inhibition of trypsin, chymotrypsin,
plasmin, cathepsin G, and human leukocyte elastase. See, for
example, Petersen et al., Eur. J. Biochem. 235:310-316, 1996. In a
typical procedure, the inhibitory activity of a test compound is
measured by incubating the test compound with the proteinase, then
adding an appropriate substrate, typically a chromogenic peptide
substrate. See, for example, Norris et al. (Biol. Chem.
Hoppe-Seyler 371:37-42, 1990). Various concentrations of the
inhibitor are incubated in the presence of trypsin, plasmin, and
plasma kallikrein in a low-salt buffer at pH 7.4, 25.degree. C.
After 30 minutes, the residual enzymatic activity is measured by
the addition of a chromogenic substrate (e.g., S2251
(D-Val-Leu-Lys-Nan) or S2302 (D-Pro-Phe-Arg-Nan), available from
Kabi, Stockholm, Sweden) and a 30-minute incubation. Inhibition of
enzyme activity is indicated by a decrease in absorbance at 405 nm
or fluorescence Em at 460 nm. From the results, the apparent
inhibition constant K.sub.i is calculated. When a serine protease
is prepared as an active precursor (e.g., comprising N-terminal
residues 1-109 of SEQ ID NO:2), it is activated by cleavage with a
suitable protease (e.g., furin (Steiner et al., J. Biol. Chem.
267:23435-23438, 1992)) prior to assay. Assays of this type are
well known in the art. See, for example, Lottenberg et al.,
Thrombosis Research 28:313-332, 1982; Cho. et al., Biochem.
0.23:644-650, 1984; Foster et al. Biochem. 26:7003-7011, 1987). The
inhibition of coagulation factors (e.g., factor VIIa, factor Xa)
can be measured using chromogenic substrates or in conventional
coagulation assays (e.g., clotting time of normal human plasma;
Dennis et al., J. Biol. Chem. 270:25411-25417, 1995).
[0117] Blood coagulation and chromogenic assays, which can be used
to detect both procoagulant, anticoagulant, and thrombolytic
activities, are known in the art. For example, pro- and
anticoagulant activities can be measured in a one-stage clotting
assay using platelet-poor or factor-deficient plasma (Levy and
Edgington, J. Exp. Med. 151:1232-1243, 1980; Schwartz et al., J.
Clin. Invest. 67:1650-1658, 1981). As disclosed by Anderson et al.
(Proc. Natl. Acad. Sci. USA 96:11189-11193, 1999), the effect of a
test compound on platelet activation can be determined by a change
in turbidity, and the procoagulant activity of activated platelets
can be determined in a phospholipid-dependent coagulation assay.
Activation of thrombin can be determined by hydrolysis of peptide
p-nitroanilide substrates as disclosed by Lottenberg et al.
(Thrombosis Res. 28:313-332, 1982). Other procoagulant,
anticoagulant, and thrombolytic activities can be measured using
appropriate chromogenic substrates, a variety of which are
available from commercial suppliers. See, for example, Kettner and
Shaw, Methods Enzymol. 80:826-842, 1981.
[0118] Anti-microbial activity of proteins is evaluated by
techniques that are known in the art. For example, anti-microbial
activity can be assayed by evaluating the sensitivity of microbial
cell cultures to test agents and by evaluating the protective
effect of test agents on infected mice. See, for example, Musiek et
al., Antimicrob. Agents Chemothr. 3:40, 1973. Antiviral activity
can also be assessed by protection of mammalian cell cultures.
Known techniques for evaluating anti-microbial activity include,
for example, Barsum et al., Eur. Respir. J. 8:709-714, 1995;
Sandovsky-Losica et al., J. Med. Vet. Mycol (England) 28:279-287,
1990; Mehentee et al., J. Gen. Microbiol (England) 135(:2181-2188,
1989; and Segal and Savage, J. Med. Vet. Mycol. 24:477-479, 1986.
Assays specific for anti-viral activity include, for example, those
described by Daher et al., J. Virol. 60:1068-1074, 1986.
[0119] The assays disclosed above can be modified by those skilled
in the art to detect the presence of agonists and antagonists of a
selected protein of interest.
[0120] Expression of a polynucleotide encoding a protein of
interest in animals provides models for further study of the
biological effects of overproduction or inhibition of protein
activity in vivo. Polynucleotides and antisense polynucleotides can
be introduced into test animals, such as mice, using viral vectors
or naked DNA, or transgenic animals can be produced.
[0121] One in vivo approach for assaying proteins of the present
invention utilizes viral delivery systems. Exemplary viruses for
this purpose include adenovirus, herpesvirus, retroviruses,
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 acids. For
review, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and
Douglas and Curiel, Science & Medicine 4:44-53, 1997. The
adenovirus system offers several advantages. Adenovirus can (i)
accommodate relatively; large DNA inserts; (ii) be grown to
high-titer; (iii) infect a broad range of mammalian cell types; and
(iv) be used with many different promoters including ubiquitous,
tissue specific, and regulatable promoters. Because adenoviruses
are stable in the bloodstream, they can be administered by
intravenous injection.
[0122] 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. 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 (e.g., the human 293 cell line). When
intravenously administered to intact animals, adenovirus primarily
targets the liver. If the adenoviral delivery system has an E1 gene
deletion, the virus cannot replicate in the host cells. However,
the host's tissue (e.g., liver) will express and process (and, if a
signal sequence is present, secrete) the heterologous protein.
Secreted proteins will enter the circulation in the highly
vascularized liver, and effects on the infected animal can be
determined.
[0123] An alternative method of gene delivery comprises removing
cells from the body and introducing a vector into the cells as a
naked DNA plasmid. The transformed cells are then re-implanted in
the body. Naked DNA vectors are introduced into host cells by
methods known in the art, including transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun, or use of a DNA vector
transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988;
Wu et al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang,
Meth. Cell Biol. 43:353-365, 1994.
[0124] Transgenic mice, engineered to express a gene encoding a
protein of interest, and mice that exhibit a complete absence of
gene function, referred to as "knockout mice" (Snouwaert et al.,
Science 257:1083, 1992), can also be generated (Lowell et al.,
Nature 366:740-742, 1993). These mice can be employed to study the
gene of interest and the protein encoded thereby in an in vivo
system. Transgenic mice are particularly useful for investigating
the role of proteins in early development in that they allow the
identification of developmental abnormalities or blocks resulting
from the over- or underexpression of a specific factor. See also,
Maisonpierre et al., Science 277:55-60, 1997 and Hanahan, Science
277:48-50, 1997. Preferred promoters for transgenic expression
include promoters from metallothionein and albumin genes. As
disclosed above, the human sequences provided herein can be used to
clone orthologous polynucleotides, which may be preferred for use
in generating transgenic and knockout animals.
[0125] Antisense methodology can be used to inhibit gene
transcription to examine the effects of such inhibition in vivo.
Polynucleotides that are complementary to a segment of a
protein-encoding polynucleotide are designed to bind to the
encoding mRNA and to inhibit translation of such mRNA. Such
antisense oligonucleotides can also be used to inhibit expression
of protein-encoding genes in cell culture.
[0126] Biological activities of test proteins can also be measured
in animal models by administering the test protein, by itself or in
combination with other agents, including other proteins. Using such
models facilitates the assay of the test protein by itself or as an
inhibitor or modulator of another agent, and also facilitates the
measurement of combinatorial effects of bioactive compounds.
[0127] Anti-inflammatory activity can be tested in animal models of
inflammatory disease. For example, animal models of psoriasis
include the analysis of histological alterations in adult mouse
tail epidermis (Hofbauer et al, Brit. J. Dermatol. 118:85-89, 1988;
Bladon et al., Arch Dermatol. Res. 277:121-125, 1985). In this
model, anti-psoriatic activity is indicated by the induction of a
granular layer and orthokeratosis in areas of scale between the
hinges of the tail epidermis. Typically, a topical ointment
comprising a test compound is applied daily for seven consecutive
days, then the animal is sacrificed, and tail skin is examined
histologically. An additional model is provided by grafting
psoriatic human skin to congenitally athymic (nude) mice (Krueger
et al., J. Invest. Dermatol. 64:307-312, 1975). Such grafts have
been shown to retain the characteristic histology for up to eleven
weeks. As in the mouse tail model, the test composition is applied
to the skin at predetermined intervals for a period of one to
several weeks, at which time the animals are sacrificed and the
skin grafts examined histologically. A third model has been
disclosed by Fretland et al. (Inflammation 14:727-739, 1990).
Briefly, inflammation is induced in guinea pig epidermis by
topically applying phorbol ester (phorbol-12-myristate-13-acetate;
PMA), typically at ca. 2 g/ml in acetone, to one ear and vehicle to
the contralateral ear. Test compounds are applied concurrently with
the PMA, or may be given orally. Histological analysis is performed
at 96 hours after application of PMA. This model duplicates many
symptoms of human psoriasis, including edema, inflammatory cell
diapedesis and infiltration, high LTB.sub.4 levels and epidermal
proliferation.
[0128] Cerebral ischemia can be studied in a rat model as disclosed
by Relton et al. (ibid.) and Loddick et al. (ibid.).
[0129] The effect of a test protein on primordial endothelial cells
in angiogenesis can be assayed in the chick chorioallantoic
membrane angiogenesis assay (Leung, Science 246:1306-1309, 1989;
Ferrara, Ann. NY Acad. Sci. 752:246-256, 1995). Briefly, a small
window is cut into the shell of an eight-day old fertilized egg,
and a test substance is applied to the chorioallantoic membrane.
After 72 hours, the membrane is examined for neovascularization.
Embryo microinjection of early stage quail (Coturnix coturnix
japonica) embryos can also be used (Drake et al., Proc. Natl. Acad.
Sci. USA 92:7657-7661, 1995). Briefly, a solution containing the
protein is injected into the interstitial space between the
endoderm and the splanchnic mesoderm of early-stage embryos using a
micropipette and micromanipulator system. After injection, embryos
are placed ventral side down on a nutrient agar medium and
incubated for 7 hours at 37.degree. C. in a humidified CO.sub.2/air
mixture (10%/90%). Vascular development is assessed by microscopy
of fixed, whole-mounted embryos and sections.
[0130] Stimulation of coronary collateral growth can be measured in
known animal models, including a rabbit model of peripheral limb
ischemia and hind limb ischemia and a pig model of chronic
myocardial ischemia (Ferrara et al., Endocrine Reviews 18:4-25,
1997). Test proteins are assayed in the presence and absence of
VEGF and basic FGF to test for combinatorial effects. These models
can be modified by the use of adenovirus or naked DNA for gene
delivery as disclosed in more detail above, resulting in local
expression of the test protein(s).
[0131] Angiogenic activity can also be tested in a rodent model of
corneal neovascularization as disclosed by Muthukkaruppan and
Auerbach, Science 205:1416-1418, 1979, wherein a test substance is
inserted into a pocket in the cornea of an inbred mouse. For use in
this assay, proteins are combined with a solid or semi-solid,
biocompatible carrier, such as a polymer pellet. Angiogenesis is
followed microscopically. Vascular growth into the corneal stroma
can be detected in about 10 days.
[0132] Angiogenic activity can also be tested in the hampster cheek
pouch assay (Hockel et al., Arch. Surg. 128:423429, 1993). A test
substance is injected subcutaneiously into the cheek pouch, and
after five days the pouch is examined under low magnification to
determine the extent of neovascularization. Tissue sections can
also be examined histologically.
[0133] Induction of vascular permeability is measured in assays
designed to detect leakage of protein from the vasculature of a
test animal (e.g., mouse or guinea pig) after administration of a
test compound (Miles and Miles, J. Physiol. 118:228-257, 1952; Feng
et al., J. Exp. Med. 183:1981-1986, 1996).
[0134] 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.
[0135] Inhibition of tumor metastasis can be assessed in mice into
which cancerous cells or tumor tissue have been introduced by
implantation or injection (e.g., Brown, Advan. Enzyme Regul.
35:293-301, 1995; Conway et al., Clin. Exp. Metastasis 14:115-124,
1996).
[0136] Effects on 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.
[0137] The invention further provides polypeptides that comprise an
epitope-bearing portion of a protein as shown in SEQ ID NO:M,
wherein M is selected form the group consisting of 2, 5, and 8. An
"epitope" is a region of a protein to which an antibody can bind.
See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA
81:3998-4002, 1984. Epitopes can be linear or conformational, the
latter being composed of discontinuous regions of the protein that
form an epitope upon folding of the protein. Linear epitopes are
generally at least 6 amino acid residues in length. Relatively
short synthetic peptides that mimic part of a protein sequence are
routinely capable of eliciting an antiserum that reacts with the
partially mimicked protein. See, for example, Sutcliffe et al.,
Science 219:660-666, 1983. Antibodies that recognize short, linear
epitopes are particularly useful in analytic and diagnostic
applications that employ denatured protein, such as Western
blotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979).
Antibodies to short peptides may also recognize proteins in native
conformation and will thus be useful for monitoring protein
expression and protein isolation, and in detecting proteins in
solution, such as by ELISA or in immunoprecipitation studies.
[0138] Antigenic, epitope-bearing polypeptides of the present
invention are useful for raising antibodies, including monoclonal
antibodies, that specifically bind to the corresponding protein.
Antigenic, epitope-bearing polypeptides contain a sequence of at
least six, preferably at least nine, more preferably from 15 to
about 30 contiguous amino acid residues of a protein. Within
certain embodiments of the invention, the polypeptides comprise 40,
50, 100, or more contiguous residues of a protein as shown in SEQ
ID NO:M, up to the entire predicted mature protein or the primary
translation product. It is preferred that the amino acid sequence
of the epitope-bearing polypeptide is selected to provide
substantial solubility in aqueous solvents, that is the sequence
includes relatively hydrophilic residues, and hydrophobic residues
are substantially avoided. Table 7 lists preferred hexapeptides for
use as antigens. Within Table 3, each the amino termini of the
hexapeptides are specified. Those skilled in the art will recognize
that longer polypeptides comprising these hexapeptides can also be
used and will often be preferred.
3 TABLE 3 Protein Hexapeptide N-termini Ztth1 49 48 91 2 90 Zsig96
45 112 59 58 57 Zcyto27 93 92 108 45 91
[0139] As used herein, the term "antibodies" includes polyclonal
antibodies, monoclonal antibodies, antigen-binding fragments
thereof such as F(ab').sub.2 and Fab fragments, single chain
antibodies, and the like, including genetically engineered
antibodies. Non-human antibodies can be humanized by grafting only
non-human CDRs onto human framework and constant regions, or by
incorporating the entire non-human variable domains (optionally
"cloaking" them with a human-like surface by replacement of exposed
residues, wherein the result is a "veneered" antibody). In some
instances, humanized antibodies may retain non-human residues
within the human variable region framework domains to enhance
proper binding characteristics. Through humanizing antibodies,
biological half-life may be increased, and the potential for
adverse immune reactions upon administration to humans is reduced.
One skilled in the art can generate humanized antibodies with
specific and different constant domains (i.e., different Ig
subclasses) to facilitate or inhibit various immune functions
associated with particular antibody constant domains.
[0140] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to an immunogenic polypeptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
an immobilized or labeled polypeptide). Human antibodies can be
produced in transgenic, non-human animals that have been engineered
to contain human immunoglobulin genes as disclosed in WIPO
Publication WO 98/24893. It is preferred that the endogenous
immunoglobulin genes in these animals be inactivated or eliminated,
such as by homologous recombination.
[0141] Antibodies are defined to be specifically binding if they
bind to a target polypeptide with an affinity at least 10-fold
greater than the binding affinity to control (non-target)
polypeptide. It is preferred that the antibodies exhibit 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
affinity of a monoclonal antibody can be readily determined by one
of ordinary skill in the art (see, for example, Scatchard, Ann. NY
Acad. Sci. 51-660 672, 1949).
[0142] Methods for preparing polyclonal and monoclonal antibodies
are well known in the art (see for example, Hurrell, J. G. R., Ed.,
Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC
Press, Inc., Boca Raton, Fla., 1982). As would be evident to one of
ordinary skill in the art, polyclonal antibodies can be generated
from a variety of warm-blooded animals such as horses, cows, goats,
sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity
of a polypeptide immunogen may 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 a polypeptide of
interest 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.
[0143] A variety of assays known to those skilled in the art can be
utilized to detect antibodies that specifically bind to a
polypeptide of interest. Exemplary assays are described in detail
in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold
Spring Harbor Laboratory Press, 1988. Representative examples of
such assays include concurrent immunoelectrophoresis,
radio-immunoassays, radio-immunoprecipitations, enzyme-linked
immunosorbent assays (ELISA), dot blot assays, Western blot assays,
inhibition or competition assays, and sandwich assays.
[0144] Antibodies can be used, for example, to isolate target
polypeptides by affinity purification, for diagnostic assays for
determining circulating or localized levels of target polypeptides,
for tissue typing, for cell sorting, for screening expression
libraries; for generating anti-idiotypic antibodies, and as
neutralizing antibodies or as antagonists to block protein activity
in vitro and in vivo.
[0145] Additionally, antibodies generated to the MSP proteins
described herein can be used, either individually or in
combination, to monitor these proteins in bodily fluids. In
particular, correlating changes in the level of expression of the
MSP proteins with changes in metabolism, or progression of disease
would be useful in studying metabolism disorders or monitoring the
improvement or decline of diseases.
[0146] Polynucleotides and polypeptides of the present invention
will additionally find use as educational tools as a laboratory
practicum kits for courses related to genetics and molecular
biology, protein chemistry and antibody production and analysis.
Due to their unique polynucleotide and polypeptide sequences
molecules of MSP can be used as standards or as "unknowns" for
testing purposes. For example, MSP polynucleotides can be used as
aids, such as, for example, to teach a student how to prepare
expression constructs for bacterial, viral, and/or mammalian
expression, including fusion constructs, wherein MSP are the genes
to be expressed; for determining the restriction endonuclease
cleavage sites of the polynucleotides; determining mRNA and DNA
localizations of MSP 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.
[0147] MSP polypeptides can be used educationally as aids to teach
preparation of antibodies; identifying proteins by Western
blotting; protein purification; determining the weight of expressed
MSP 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., receptor binding, signal transduction,
proliferation, and differentiation) in vitro and in vivo. MSP
polypeptides can also be used to teach analytical skills such as
mass spectrometry, circular dichroism to determine conformation, in
particular the locations of the disulfide bonds, x-ray
crystallography to determine the three-dimensional structure in
atomic detail, nuclear magnetic resonance spectroscopy to reveal
the structure of proteins in solution. For example, a kit
containing the MSP can be given to the student to analyze. Since
the amino acid sequences would be known by the professor, the
proteins can be given to the student as a test to determine the
skills or develop the skills of the student, the teacher would then
know whether or not the student has correctly analyzed the
polypeptides. Since every polypeptide is unique, the educational
utility of each MSP would be unique unto itself.
[0148] The antibodies which bind specifically to a MSP can be used
as a teaching aid to instruct students how to prepare affinity
chromatography columns to purify MSP, cloning and sequencing the
polynucleotide that encodes an antibody and thus as a practicum for
teaching a student how to design humanized antibodies. The MSP
gene,s polypeptides or antibodies would then be packaged by reagent
companies and sold to universities 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 MSP genes, polypeptides or
antibodies are considered within the scope of the present
invention.
[0149] The present invention also provides reagents for use in
diagnostic and therapeutic applications. Such reagents include
polynucleotide probes and primers; antibodies, including antibody
fragments, single-chain antibodies, and other genetically
engineered forms; soluble receptors and other polypeptide binding
partners; and the proteins of the invention themselves, including
fragments thereof. Those skilled in the art will recognize that
diagnostic reagents will commonly be labeled to provide a
detectable signal or other second function. Thus, polypeptides,
antibodies, receptors, and other binding partners disclosed herein
can be directly or indirectly conjugated to drugs, toxins,
radionuclides, enzymes, enzyme substrates, cofactors, inhibitors,
fluorescent markers, chemiluminescent markers, magnetic particles,
and the like, and these conjugates used for in vivo diagnostic or
therapeutic applications. Cytotoxic molecules, for example, can be
directly or indirectly attached to the binding partner (e.g., by
chemical coupling or as a fusion protein), and include bacterial or
plant toxins (e.g., diphtheria toxin, Pseudomonas exotoxin, ricin,
saporin, abrin, and the like); therapeutic radionuclides (e.g.,
iodine-131, rhenium-188 or yttrium-90) which can be directly
attached to a polypeptide or antibody or indirectly attached
through means of a chelating moiety; and cytotoxic drugs (e.g.,
adriamycin). Methods for preparing labeled reagents are known in
the art. Within an alternative embodiment, the detectable signal or
other function can be provided by a second member of a
complement-anticomplement pair, which second member binds to the
diagnostic reagent. For example, a first (unlabeled) antibody can
be used to bind to a cell-surface polypeptide, after which a
second, labeled antibody which binds to the first antibody is
added. Other complement-anticomplement pairs are known in the art
and include biotin/streptavidin.
[0150] Diagnostic reagents as disclosed herein can be used in vivo
or in vitro. In vitro diagnostic assays include assays of tissue
and fluid samples. Assays for protein in serum, for example, may be
used to detect metabolic abnormalities characterized by over- or
under-production of the protein, such as cancers, immune system
abnormalities, infections, organ failure, metabolic imbalances,
inborn errors of metabolism and other disease states. Proteins of
the present invention can also be used in the detection of
circulating autoantibodies, which are indicative of autoimmune
disorders. Those skilled in the art will recognize that conditions
related to protein underexpression or overexpression may be
amenable to treatment by therapeutic manipulation of the relevant
protein level(s). Protein, in serum can be quantitated by known
methods known in the art, which include the use of antibodies in a
variety of formats. Non-antibody binding partners, such as
ligand-binding receptor fragments (commonly referred to as "soluble
receptors") can also be used.
[0151] In general, diagnostic methods employing oligonucleotide
probes or primers comprise the steps of (a) obtaining a genetic
sample from a patient; (b) incubating the genetic sample with an
oligonucleotide probe or primer as disclosed above, under
conditions wherein the probe or primer will hybridize to a
complementary polynucleotide sequence, to produce a first reaction
product; and (c) comparing the first reaction product to a control
reaction product. A difference between the first reaction product
and the control reaction product is indicative of a genetic
abnormality in the patient. Genetic samples for use within such
methods include genomic DNA, cDNA, and RNA. Suitable assay methods
in this regard include molecular genetic techniques known to those
in the art, such as restriction fragment length polymorphism (RFLP)
analysis, short tandem repeat (STR) analysis employing PCR
techniques, ligation chain reaction (Barany, PCR Methods and
Applications 1:5-16, 1991), ribonuclease protection assays, and
other genetic linkage analysis techniques known in the art
(Sambrook et al., ibid.; Ausubel et. al., ibid.; A. J. Marian,
Chest 108:255-65, 1995). Ribonuclease protection assays (see, e.g.,
Ausubel et al., ibid., ch. 4) comprise the hybridization of an RNA
probe to a patient RNA sample, after which the reaction product
(RNA-RNA hybrid) is exposed to RNase. Hybridized regions of the RNA
are protected from digestion. Within PCR assays, a patient genetic
sample is incubated with a pair of oligonucleotide primers, and the
region between the primers is amplified and recovered. Changes in
size, amount, or sequence of recovered product are indicative of
mutations in the patient. Another PCR-based technique that can be
employed is single strand conformational polymorphism (SSCP)
analysis (Hayashi, PCR Methods and Applications 1:34-38, 1991).).
Chromosomal localization data can be used to correlate MSP gene
locations with known genetic disorders using, for example, the
OMIM.TM. Database, Johns Hopkins University, 2000
(http:/Hwww.ncbi.nlm.nih.gov/entrez/query.fcgi ?db=OMIM).
[0152] Polynucleotides of the present invention, including
fragments thereof, can also be used for radiation hybrid mapping, a
somatic cell genetic technique developed for constructing
high-resolution, contiguous maps of mammalian chromosomes (Cox et
al., Science 250:245-50, 1990). Partial or full knowledge of a
gene's sequence allows the design of PCR primers suitable for use
with chromosomal radiation hybrid mapping panels. Commercially
available radiation hybrid mapping panels which cover the entire
human genome, such as the Stanford G3 RH Panel and the GeneBridge 4
RH Panel (Research Genetics, Inc., Huntsville, Ala.), are
available. These panels enable rapid, PCR-based chromosomal
localizations and ordering of genes, sequence-tagged sites (STSs),
and other nonpolymorphic and polymorphic markers within a region of
interest, allowing the establishment of directly proportional
physical distances between newly discovered genes of interest and
previously mapped markers. The precise knowledge of a gene's
position can be useful for a number of purposes, including: 1)
determining if a sequence is part of an existing contig and
obtaining additional surrounding genetic sequences in various
forms, such as YACs BACs or cDNA clones; 2) providing a possible
candidate gene for an inheritable disease which shows linkage to
the same chromosomal region; and 3) cross-referencing model
organisms, such as mouse, which may aid in determining what
function a particular gene might have.
[0153] If a mammal has an insufficiency of a protein of interest
(due to, for example, a mutated or absent gene), the corresponding
wild-type gene can be introduced into the cells of the mammal. In
one embodiment, a gene encoding a protein of interest is introduced
into the animal using a viral vector. Such vectors include an
attenuated or defective DNA virus, such as, but not limited to,
herpes simplex virus (HSV), papillomavirus, Epstein Barr virus
(EBV), adenovirus, adeno-associated virus (AAV), and the like.
Defective viruses, which entirely or almost entirely lack viral
genes, are preferred. A defective virus is not infective after
introduction into a cell. Use of defective viral vectors allows for
administration to cells in a specific, localized area, without
concern that the vector can infect other cells. Examples of
particular vectors include, but are not limited to, a defective
herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as
the vector described by Stratford-Perricaudet et al. (J. Clin.
Invest. 90:626-30, 1992); and a defective adeno-associated virus
vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et
al., J. Virol. 63:3822-28, 1989).
[0154] Within another embodiment, a gene of interest is introducted
into an animal by liposome-mediated transfection ("lipofection")
essentially as disclosed above. Lipofection can be used to
introduce exogenous genes into specific organs.
[0155] A gene of interest can also be introduced into an animal for
gene therapy as a naked DNA plasmid using the methods disclosed
above.
[0156] In another embodiment, polypeptide-toxin fusion proteins or
antibody/fragment-toxin fusion proteins may be used for targeted
cell or tissue inhibition or ablation, such as in cancer therapy.
Of particular interest in this regard are conjugates of an MSP
protein and a cytotoxin, which can be used to target the cytotoxin
to a tumor or other tissue that is undergoing undesired
angiogenesis or neovascularization.
[0157] In another embodiment, MSP-cytokine fusion proteins or
antibody/fragment-cytokine fusion proteins may be used for
enhancing in vitro cytotoxicity (for instance, that mediated by
monoclonal antibodies against tumor targets) and for enhancing in
vivo killing of target tissues (for example, blood and bone marrow
cancers). See, generally, Hornick et al., Blood 89:4437-4447,
1997). In general, cytokines are toxic if administered
systemically. The described fusion proteins enable targeting of a
cytokine to a desired site of action, such as a cell having binding
sites for an MSP protein, thereby providing an elevated local
concentration of cytokine. Polypeptides, antibodies, or receptors
target an undesirable cell or tissue (e.g., a tumor), and the fused
cytokine mediates improved target cell lysis by effector cells.
Suitable cytokines for this purpose include, for example,
interleukin-2 and granulocyte-macrophage colony-stimulating factor
(GM-CSF).
[0158] In another embodiment, polypeptide-toxin fusion proteins or
other binding partner-linked toxins may be used for targeted cell
or tissue inhibition or ablation (for instance, to treat cancer
cells or tissues). Target cells (i.e., those displaying a receptor
for a polypeptide of interest) bind the polypeptide-toxin
conjugate, which is then internalized, killing the cell. The
effects of receptor-specific cell killing (target ablation) are
revealed by changes in whole animal physiology or through
histological examination. Thus, ligand-dependent, receptor-directed
cyotoxicity can be used to enhance understanding of the
physiological significance of a protein ligand. A preferred such
toxin is saporin. Mammalian cells have no receptor for saporin,
which is non-toxic when it remains extracellular. Alternatively, if
the polypeptide of interest has multiple functional domains (i.e.,
an activation domain or a ligand binding domain, plus a targeting
domain), a fusion protein including only the targeting domain may
be suitable for directing a detectable molecule, a cytotoxic
molecule or a complementary molecule to a cell or tissue type of
interest. In instances where the domain-only fusion protein
includes a complementary molecule, the anti-complementary molecule
can be conjugated to a detectable or cytotoxic molecule. Such
domain-complementary molecule fusion proteins thus represent a
generic targeting vehicle for cell- or tissue-specific delivery of
generic anti-complementary-detectable/cytotoxic molecule
conjugates.
[0159] The bioactive conjugates described herein can be delivered
intravenously, intraarterially or intraductally, or may be
introduced locally at the intended site of action.
[0160] For pharmaceutical use, the proteins of the present
invention are formulated according to conventional methods. Routes
of delivery include topical, mucosal, and parenteral, the latter
including intravenous and subcutaneous delivery. Intravenous
administration will be by bclus injection or infusion over a
typical period of one to several hours. In general, pharmaceutical
formulations will include a protein of the present invention in
combination with a pharmaceutically acceptable vehicle, such as
saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients, diluents,
fillers, emulsifiers, preservatives, solubilizers, buffering
agents, wetting agents, stabilizers colorings, penetration
enhancers, albumin to prevent protein loss on vial surfaces, etc.
Topical formulations are typically provided as liquids, ointments,
salves, gels, emulsions and the like. Methods of formulation are
well known in the art and are disclosed, for example, in Remington:
The Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing
Co., Easton, Pa., 19th ed., 1995. Therapeutic doses will be
determined by the clinician according to accepted standards, taking
into account the nature and severity of the condition to be
treated, patient traits, etc. Proteins of the present invention
will generally be formulated to provide a dose of from 0.01 .mu.g
to 100 mg per kg patient weight per day, more commonly from 0.1
.mu.g to 10 mg/kg/day, still more commonly from 0.1 .mu.g to 1.0
mg/kg/day. Determination of dose is within the level of ordinary
skill in the art. The proteins may be administered for acute
treatment, over one week or less, often over a period of one to
three days or may be used in chronic treatment, over several months
or years. In general, a therapeutically effective amount is an
amount sufficient to produce a clinically significant change in the
targeted condition.
[0161] Within the laboratory research field, the proteins of the
present invention can be used as molecular weight standards, or as
standards in the analysis of cell phenotype, and as reagents for
the study of cells, receptors, and other binding molecules. Such
reagents will generally further comprise a second moiety, such as a
label, binding partner, or toxin, that facilitates the detection of
the protein when bound to its target. Many such systems are known
in the art and are summarized above. Receptors and other
cell-surface binding sites for proteins of the present invention
can be identified by exposing a population of cells to a labeled
protein under physiologic conditions, whereby the protein binds to
the surface of the cell. Cells bearing receptors for a protein of
interest can also be identified using the protein joined to a
toxin, whereby receptor-bearing cells are killed by the toxin.
[0162] MSP proteins and antagonists thereof can be used as
standards in assays of protein and protein inhibitors in both
clinical and research settings. Such assays can comprise any of a
number of standard formats, include radioreceptor assays and
ELISAs. Protein standards can be prepared in labeled form using a
radioisotope, enzyme, fluorophore, or other compound that produces
a detectable signal. The proteins can be packaged in kit form, such
kits comprising one or more vials containing the MSP protein and,
optionally, a diluent, an antibody, a labeled binding protein, etc.
Assay kits can be used in the research laboratory to detect protein
and inhibitor activities produced by cultured cells or test
animals.
[0163] Proteins of the present invention may also be used as
protein and amino acid supplements, including hydrolysates.
Specific uses in this regard include use as animal feed supplements
and as cell culture components. Protein rich in a particular amino
acid can be used as a source of that amino acid.
[0164] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
[0165] A protein of the present invention ("MSP") 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 MSP
sequences.
[0166] 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 SmaI site just
downstream of the MBP-His.sub.6-thrombin site coding sequences. The
MSP 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 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.
[0167] 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.
[0168] E. coli strain BL21 is used for expression of MSP. Cells are
transformed by electroporation and grown on minimal glucose plates
containing casamino acids and ampicillin.
[0169] 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
[0170] Larger scale cultures of MSP 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
[0171] For larger-scale protein preparation, 500-ml cultures of E.
coli BL21 expressing the MSP-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.
[0172] Recombinant MSP 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-MEP
antibody.
Example 4
[0173] An expression plasmid containing all or part of a
polynucleotide encoding MSP is constructed via homologous
recombination. An MSP coding 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 MSP.
[0174] 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. Plamid 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.
[0175] 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+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 ml 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 .mu.l H.sub.2O.
[0176] Transformation of electrocompetent E. coli host cells
(Electromax DH10.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 ml 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).
[0177] Individual clones harboring the correct expression construct
for MSP are identified by restriction digest to verify the presence
of the MSP 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/MSP.
[0178] Recombinant protein is produced in BHK cells transfected
with pZMP6/MSP. 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/MSP 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-propanimini-
um-trifluoroacetate and the neutral lipid dioleoyl
phosphatidylethanolamin- e in membrane-filtered water
(Lipofectamine.TM. Reagent; Life Technologies; Garithersburg, Md.),
in serum free (SF) media (DMEM supplemented with 10 mg/ml
transferrin, 5 mg/ml 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.
[0179] 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.
* * * * *
References