JP2003521230A - Exocytosis pathway proteins and methods of use - Google Patents

Abstract

  (57) [Summary] The present invention relates to novel polypeptides, such as Exo proteins, and related molecules that affect or associate with exocytosis, and nucleic acid molecules that encode those polypeptides. In addition, vectors and host cells containing those nucleic acid sequences, chimeric polypeptide molecules containing the polypeptide of the present invention fused to a variant polypeptide sequence, antibodies that bind to the polypeptide of the present invention, and polypeptides of the present invention Methods for preparing are also provided. Further, the present invention provides methods for identifying novel compositions that mediate Exo bioactivity, and the use of those compositions in the diagnosis and treatment of disease.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to molecules involved in the exocytosis pathway, and more specifically,
It relates to novel polypeptides related to exosite linear function proteins, nucleic acids and antibodies. The present invention further provides proteins associated with the exocytosis pathway,
The present invention relates to use in a method for identifying a candidate substance that regulates exocytosis.

BACKGROUND OF THE INVENTION In eukaryotic cells, proteins destined for the plasma membrane or extracellular space are delivered along the secretory pathway. It consists of a series of sequential, vesicle-mediated transport steps, each of which requires specific targeting of the transport vesicle to the appropriate acceptor membrane and subsequent fusion of the vesicle with the acceptor membrane. And In this way, the protein to be secreted by the cell migrates into the endoplasmic reticulum and then through the Golgi complex. The protein is divided in secretory vesicles in the trans-Golgi network, and these vesicles then fuse with the plasma membrane. This last membrane fusion event, known as exocytosis, results in the release of vesicle contents into the extracellular space and uptake of vesicle membrane lipids and proteins into the plasma membrane.

Exocytosis can be divided into two classes: constitutive (constit
utive) and regulated. In constitutive exocytosis, secretory vesicles fuse with the plasma membrane immediately after generation; in regulatory exocytosis, secretory vesicles accumulate in the cytoplasm and fuse only when they receive the appropriate signal I do. All eukaryotic cells exhibit constitutive exocytosis.

The basic purpose of exocytosis is to deliver lipids and proteins to the plasma membrane to release the contents of vesicles from cells, but various cell types utilize this mechanism. Perform their own physiological role. Some examples of different functions of exocytosis in different cell types are listed in Table 1.

[Table 1]

[Table 2]

It should be noted that exocytosis (ie, the fusion of vesicles with the plasma membrane) is not just the end of the secretory pathway, but can also involve vesicles not derived from the endoplasmic reticulum. is there. For example, transcytosis occurs in polarized cells and transports from one pole of the cell to the budding of endocytic vesicles to another (
Often via the endosome) and subsequent endocytic fusion. In breast cells, transcytosis is used for uptake of antibodies from the blood and subsequent secretion into milk. Similarly, some vesicles undergo exo / endocytic fusion via endosomes without returning to the Golgi apparatus. The exocytosis of recycling vesicles may be either constitutive (eg transferrin receptor-containing vesicles) or regulatory (eg synaptic vesicles).

Since all cells exhibit constitutive exocytosis, regulatory secretory cells must have two types of secretory vesicles: one constitutive and the other regulatory. . Morphological studies indicate that this is so, because constitutive secretory vesicles appear small and clearly under electron microscopy, while regulatory secretory vesicles are typically more Because it appears large and opaque. Furthermore,
The two types of vesicles usually contain different substances (with the exception of mammary cells, where casein secretion occurs by both constitutive and regulatory exocytosis).

It should be noted that the cells may contain one or more types of regulatory secretory vesicles. This optimal example is found in neurons, which may have synaptic vesicles and large dense-core vesicles in addition to constitutive secretory vesicles. Some properties of the two types of neuronal regulatory secretory vesicles are displayed in Table 2. Large dense-core vesicles contain peptide neurotransmitters and they are very similar to regulatory secretory vesicles in endocrine cells. In fact, much of the information about large dense-core vesicle biogenesis and exocytosis comes from the study of adrenal chromaffin cells and their tumor cell derivatives, PC12 cells, which are both popular neuronal cells. Synaptic vesicles are clearly visible by electron microscopy, are much smaller than large dense-core vesicles, and contain fast neurotransmitters. Synaptic vesicles have evolved in animals to allow the extremely rapid point-to-point communication required for brain function. Recently, synaptic vesicles have been found in endocrine cells such as adrenal chromaffin cells and pancreatic β-cells. These vesicles also appear to contain fast neurotransmitters, but their physiological role is unclear.

[0008]

[Table 3] Abbreviations used: GABA, λ-aminobutyric acid; ACh, acetylcholine; VIP, vasoactive intestinal peptide

The more information gathered about exocytosis, the easier it will be to manipulate exocytosis. Moreover, the more information collected, the easier it will be to diagnose and treat diseases involved in exocytosis. For example, the release of inflammatory mediators from mast cells leads to various diseases including asthma. Treatments for allergies remain limited to blockade of individual mediators released from mast cells (antihistamines), nonspecific anti-inflammatory agents such as steroids and mast cell stabilizers, but they are symptomatic of allergies. It is only marginally effective in suppressing the.

Similarly, the Chedial-Higashi syndrome (CHS) is a rare autosomal recessive disease in which neutrophils, monocytes and lymphocytes contain giant cytoplasmic granules.
Similar diseases have been described in mice, mink, cattle, cats and orcas, C
The mouse homologue of HS (designated beige or bg) is the best characterized. Perou et al., J. Biol. Chem., Both incorporated herein by reference.
272 (47): 29790 (1997) and Barbosa et al., Nature 382: 262 (1996).

Therefore, it is necessary to determine the proteins involved in exocytosis.
Some insight into the process of regulated secretion at the molecular level has allowed its definition as a key regulator of G proteins. Early experiments showed that a non-hydrolyzable analog of GTP caused secretion in peritoneal mast cells (Fer
nandes, et al., Nature 312: 453 (1984)). More recently, a large body of evidence has accumulated that rab family small G proteins are involved as regulators in the fusion of secretory granules and cytoplasmic membranes during exocytosis. RabGTPases represent a distinct family of homologous proteins commonly associated with the membranes of organelles in a wide variety of cells, where they regulate specific steps of intracellular membrane trafficking (Zerial, M. and Stenmark, H., Curr. Opin.
Cell Biol. 5: 613 (1993)). An example of this is the rab3 subfamily of proteins, which have been found to have restricted expression in regulatory secretory-competent cells and are associated with synapses or secretory granules, which stimulate them. Suggested to be involved in secretory coupling (Lledo et al., Trends Neurobiol. Sci.
17: 426 (1994)). Furthermore, in rat basophil RBL, rab3d or its G
Overexpression of the TP-binding mutant (N1351) leads to a significant inhibition of IgE-mediated exocytosis (Roa, J. Immunol., 159: 2815 (1997)).

[0012] RAB3a, RAB3b, RAB3c and RAB3d constitute a subgroup of the rab family involved in regulated secretion. Rab3a is a neuron,
It has been detected in regulatory secretory cells such as endocrine and exocrine cells but not in constitutive secretory cells such as hepatocytes and lymphocytes (Fischer
von Mollard, Proc. Natl. Acad. Sci. 87: 1988-92 (1990), Takai, et al.,
Int. Rev. Cytol. 133: 187-230 (1992)). At neuromuscular synapses, rab
3a is localized in synaptic vesicles (Mizoguchi, et al., Biochem. Biophys. Re
S. Commun. 202: 1235-43 (1994)). Furthermore, Rab3a has also been detected in secretory granules of chromaffin cells and in zymogen granules in exocrine cells of the pancreatic acinar (Padfield et al., Proc. Natl. Acad. Sci. USA 89: 1656-). 60 (
1992)). Rab3a is an exchange protein for GDI and GRF (Matsui et al.,
Mol. Cell. Bio. 10: 4116-22 (1990), Burstein, ES and Macara IG, Pr.
oc. Natl. Acad. Sci. USA 89: 1154-8 (1992)) and GAP (Burstein, J. B.
iol. Chem. 266: 2689-92 (1991)) and Rabphilin (Shirataki, et al.
., Mol. Cell Biol. 13: 2061-8 (1993)) and has been shown to interact with a number of proteins. Rab3a is believed to regulate exocytosis in regulatory secretory cells. Rab3a has been cloned and is known in the art (ie, Genbank accession number (no.) M28210).

Rab3d is believed to be involved in the regulation of regulatory secretion in numerous cell types. Rab3d is preferentially expressed in adipocytes, but also in lung, spleen,
It can be found to be expressed at low levels in a wide range of tissue types, including heart and brain. Baldini, G., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 5
049-52. Rab3d is also involved in translocation of the Glut4 glucose transporter in adipocytes. Rab3d has been cloned and is known in the art, ie
Genbank access no. : AF081353.

Thus, rabs, in particular tissue / cell-specific isoforms of rabs and the proteins with which they interact, are of great pharmaceutical interest. Rab7 has been cloned and is known in the art (ie, Genbank access no. U4
4104).

Rab9 localizes to the surface of slow endosomes, where it is in vitro.
Both in vivo and in vivo, it appears to act to stimulate the transport of the mannose 6-phosphate receptor between slow endosomes and the trans-Golgi network. Recent studies suggest that this GTPase is a rate-limiting component due to transport between slow endosomes and the trans-Golgi network. Rab9 has been cloned and is known in the art (ie U44103
See).

Rab11 was identified by screening a Madin-Darby canine kidney cell cDNA library with degenerate oligonucleotides derived from conserved sequences of the Rab superfamily. Chavrier, P., et al., (199
0), Mol. Cell. Biol. 10: 6578-85. Rab of dog, human, rat and rabbit
The predicted amino acid sequences of 11 are 100% identical. This high level of conservation between species may reflect the extraordinary importance of this Rab family member. Rab
11 is localized to both constitutive and regulated secretory pathways in PC12 cells.
Ora3, a homologue of Rab11 (91% identity at the amino acid level), has been found to be associated with cholinergic synaptic vesicles derived from electrical organs of marine tissues. Although the function of Rab11 has not yet been fully determined, numerous lines of evidence suggest that it may serve to target transport vesicles of different origins to a common destination, the cytoplasmic membrane. Suggest. Northern blot analysis is performed by Rab
11 is ubiquitously expressed, indicating that it is generally more abundant in tissues with high levels of secretion. Rab11 has been cloned and is known in the art, ie Genbank access no. X56740.

Rab5 (a, b, and c) form a subgroup of the Rab protein family. They localize on the cytoplasmic surface of the cytoplasmic membrane, on early endosomes and on cytoplasmic membrane-induced clathrin-coated vesicles. Antibodies directed against Rab5a inhibit the fusion of early endosomes in vivo, suggesting that its activity is required for this process. In vivo, overexpression of wild-type and mutant Rab5a results in changes in the rate of internalization of endocytic markers and in the morphological alteration of early endosomes. These data suggest that Rab5a is a rate-limiting factor that regulates the kinetics of both lateral fusion of early endosomes and fusion of cytoplasmic membrane-derived vesicles with early endosomes. Several proteins have been identified as being associated with Rab5. For example, a 62 kDa multi-stage coil protein that specifically interacts with the GTP-bound form of Rab5 has been identified. This protein is Rabaptin-5
, Shares 42% juxtapositional identity with the previously identified effector of Rab5, and is designated Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta exhibits the 7 repeats characteristic of multi-coil proteins and is recruited by Rab5 onto endosomal membranes in a GTP-dependent manner. However, Rabaptin-5beta does not
It has the characteristic of distinguishing it from tin-5. The relative expression levels of the two proteins vary in different cell types. Rabaptin-5beta is Rabapti
GDP / G for Rabex-5 and Rab5 without heterodimerization with n-5
It forms another complex with the TP exchange factor. From cytosol to Rabaptin-5
Immunodepletion of the beta complex only partially inhibits early endosomal fusions in vitro, whereas the additional loss of the Rabapin-5 complex has a stronger inhibitory effect. Fusion activity is usually restored by the addition of Rabaptin-5 complex alone, but the best fusion potency is Rabaptin-5 and Ra.
It requires the presence of both the baptin-5beta complex. Goumier H., et a
l., (1998), EMBO J. 17 (7): 1930-1940. Rab5 has been cloned and is known in the art (ie, see Genbank Access No. M28215).

Furthermore, important proteins acting in CA ²⁺ -regulated exocytosis in neurons and endocrine cells are the vesicle protein synapstagmin, VA.
MP / synapse brevin, target membrane proteins SYNAYXINs and SNAP
-23/25 and, in addition, soluble N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF-adhesion protein (α-, β-, γ-SNA
Ps) is included. Functional evidence for the importance of membrane proteins comes from their susceptibility to specific proteolytic actions of Clostridium neurotoxins and / or genetic analysis in mice and Drosophila. Soluble factor NSF
And SNAP have been found to interact with neurotoxin substrates in the 20S complex leading to them as SNAP-passives (SNAREs). SNARE
Many proteins that form complexes contain multiple coil domains that are believed to interact predominantly through hydrophobic interactions. These described proteins function at some point in the exotic pathway, either as central members of the vesicle fusion complex or as accessory proteins involved in certain regulatory steps in the vesicle fusion cycle.

[0019] GS27 is associated with the Golgi apparatus and is believed to behave like SNARE. GS27 (against the Golgi SNARE at 27K) is identical to membrin, a protein initially involved in ER-to-Golgi transport. For SNAREs, these proteins are known to mediate vesicle transport by docking vesicles on target membranes. The cytoplasmic domain of GS27 or antibodies raised against it quantitatively inhibits ER-to-Golgi / TGN transport in vitro, acting at a step between cis / intermediate- and trans-Golgi / TGN. , Intermediate- to trans-Golgi / TGN protein transfer is SNARE
-Dependence on mediated vesicle transport and GS27 have been reported in one study to play a functional role (Lowe, SL, et al., Nature, 389: 881-884 (1997)).
Therefore, GS27 is involved in exocytosis by being involved in vesicle transport.

SNAP-23 (used interchangeably with snap-23) was first identified in a human B lymphocyte cDNA library (Ravichandran, V., et al., (19
96), J. Biol. Chem. 271: 13300-03). Subsequently, the identification of SNAP-23 in several cell types, including mast cells, was independently reported by other investigators. The primary structure of SNAP-23 is 59% identical to SNAP-25; the former contains a central cluster of cysteine residues, which is the site of palmitoylation at SNAP-25, and other SNAREs, particularly syntaxin 1,3. , And 4 are expected multi-stage coils that would be useful for coupling. SNAP-23, like SNAP-25, is mainly localized to the plasma membrane. However, recent evidence suggests that SNAP-23 translocates to the surface of secretory granules upon activation of cells in SYN.
It is suggested to form a complex with TAXIN-3 and VAMP-2 (Guo, Z.
, et al., (1998), Cell. 94: 537-48). The same study shows that the function of SNAP-23 is crucial for complex exocytosis in mast cells, which is SNAP-2.
It is suggested that trispecific antibodies may completely block secretion in permeabilized cells.
SNAP-23 has been cloned and is known in the art (ie, Genb
ank access no. See U55936).

NSF and alpha-snap were originally detected as factors required for transport through the Golgi in in vitro assays, and the yeast homologues of these proteins, sec18 and sec17, are involved in secretion in vivo. Required. Α and β-SNAP appear to be functionally degenerate in the Golgi transport assay and in the formation of the Golgi membrane-induced 20S complex. In contrast, more recent results indicate that alpha- and β-SNAP are alpha-snap
It is suggested that it has another function in regulatory exocytosis based on its ability to move synaptic tagmin from the NARE complex. Sollner, T., et al., C
ell, 75: 409-18 (1993). Alpha-snap has been cloned and is known in the art, i.e. Genbank Accession No. U39412.

Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the enzyme Saccharomyces cerevisiae. Syntaxin (T-SNARE)
Shows SNAP25 and synapse brevin / VAMP (T- and V
-Docking in synaptic vesicle exocytosis with SNARE-
It is believed to form the core of the fusion complex. Proteins that are similar to Sec1 are Drosophila melanogaster (Rop) and C. elegans eleg.
was identified in the neural system of ans (UNC18). Munc-18 / n-Sec
1 / rbSec1, a brain homolog of the yeast Sec1p protein, is thought to participate in regulating docking and fusion of synaptic vesicles. Munc-18 / n
-Sec1 / rbSec1 expression has been reported to be nervous system-specific, and numerous non-neural isoforms have been identified that are more ubiquitously expressed. Shaywitz
, DA, et al., J. Cell. Biol. 128: 769-777 (1995).

The role of Munc-18c, previously identified as the n-Sec1 / Munc-18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has been investigated in 3T3-L1 adipocytes. Lowe, SL,
et al., Nature, 389: 881-884 (1997). In these cells, Munc-
18c was preferentially associated with syntaxin4, which bound both syntaxin2 and syntaxin4 to a similar extent in vitro. In addition, SN
AP23, an adipocyte homolog of SNAP25, was associated with both syntaxin 2 and 4 in 3T3-L1 adipocytes. Over-expression of Munc-18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer was used in a viral dose-dependent manner (maximum effect, approximately 50%) as well as inhibition of insulin-stimulated glucose transport as well as insulin. It results in inhibition of sorbitol-induced glucose transport (by only about 35%), which is mediated by a pathway different from that involved. In contrast, and also in adipocytes, Munc-18b, which does not bind syntaxin 4, has no effect on glucose transport. These results implicate Munc-18c in insulin-dependent trafficking from the intracellular storage compartment of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the formation of the SNARE complex containing syntaxin4. Suggest that. Uncl8-1 has been cloned and is known in the art, i.e. Genbank accession number (no.) D63851.

Tetanus toxin inhibits neurotransmitter release by selectively blocking fusion of synaptic vesicles. Tetanus toxin has been shown to proteolytically degrade synaptic brevin II (also called VAMP-2), a synaptic vesicle-specific protein, in vitro and at nerve endings. As a target for tetanus toxin, synaptic brevin probably functions in the exotic fusion of synaptic vesicles. Synapse brevin derivative, cerbrevin (VAMP-3), which is present in all cells and tissues tested, is a membrane trafficking protein of the constitutive recycling pathway. McMahon H.T. et al., Nature, 3
64 (6435): 346-52 (1993). Like Synapse Brevin II, selbrevin is in
Proteolytically in vitro and by the light chain of tetanus toxin after transfection. These results indicate that the constitutive and regulatory vesicle pathways allow cognate proteins for membrane trafficking, presumably due to membrane fusion at the plasma membrane.
It has been shown to be used and shows greater mechanical and evolutionary similarities between these pathways than previously thought. A homologue of Vamp3, vamp2 (synapse brevin II), is localized to mast cell granules and may play a crucial role in mast cell exocytosis (Guo, Z., et al., Cell. , 94: 537-
48 (1998)). Vamp3 has been cloned and is known in the art, ie G
enbank access no. : AF26007.

Therefore, proteins involved in exocytosis, referred to herein as Exo proteins, in particular GS27, Rab3a, Rab7, Rab9, Rab11, Ra.
Those associated with b3d, Rab5, alpha-snap, unc18-1, vamp3, and snap-23 are of interest, and it is desirable to provide such proteins and related molecules. It is a further aspect of this invention to provide a recombinant nucleic acid encoding an Exo protein and expression vector as well as a host cell containing the nucleic acid encoding an Exo protein. A further aspect of this invention is to provide methods of screening for antagonists and agonists of Exo proteins, especially those that mediate exocytosis, secretion and / or vesicle trafficking.

SUMMARY OF THE INVENTION Accordingly, the present invention provides a recombinant nucleic acid encoding an Exo protein, the Exo protein having SEQ ID NOS: 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, 63, 64, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102,
103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120,
121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 147, 148, 150, 151,
152, 155, 156, 157, 158, 159, 160, 161, 162,
163, 164, 165, 166, 167, 168, 169, 170, 171,
193, 194, 195, 196, 197, 198, 199, 200, 201,
At least about 85% sequence identity with an amino acid sequence encoded by a nucleic acid comprising the first 100 nucleic acid residues of a sequence selected from the group consisting of 202, 203, 204, 205, 210, 211, and more preferably Have at least about 90% sequence identity, and most preferably at least about 95% sequence identity. Preferably, these Exo proteins are GS27, rab7, rab9, sn
ap-23, lab3a, lab11, lab3d, lab5, alpha-s
It binds to a protein selected from the group consisting of nap, unc18-1 and vamp3. Also provided are recombinant nucleic acids, which are SEQ ID NOS
: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37
, 39, 41, 43, 45, 47, 49, 51, 53, 63, 64, 86, 87
, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
, 100, 101, 102, 103, 104, 105, 106, 107, 108
, 109, 110, 111, 112, 113, 114, 115, 116, 117
, 118, 119, 120, 121, 122, 123, 124, 125, 126
127, 128, 129, 130, 131, 132, 133, 134, 135
, 136, 137, 138, 139, 140, 141, 142, 143, 147
, 148, 150, 151, 152, 155, 156, 157, 158, 159
, 160, 161, 162, 163, 164, 165, 166, 167, 168.
169, 170, 171, 193, 194, 195, 196, 197, 198.
, 199, 200, 201, 202, 203, 204, 205, 210, 211
At least about 75% sequence identity, more preferably at least about 85% sequence identity, and most preferably with a nucleic acid sequence comprising the first 100 nucleic acid residues of a sequence selected from the group consisting of and the complements thereof. Have at least about 95% sequence identity. Recombinant Exo proteins containing these nucleic acids, expression vectors and host cells are also included.

In another aspect, the invention features a recombinant Exo protein, Exo3, E
xo4, Exo5, Exo6, Exo7, Exo8, Exo9, Exo10, E
xo11, Exo12, Exo13, Exo14, Exo15, Exo16, E
xo17a, Exo17b, Exo18, Exo19, Exo20, Exo21
, Exo22, Exo23, Exo24, Exo25, Exo26, Exo27
, Exo28, Exo29, Exo30, Exo31, Exo32, Exo33
, Exo34, Exo35, Exo36, Exo37, Exo38, Exo39
, Exo40, Exo41, Exo42, Exo43, Exo44, Exo45
, Exo46, Exo47, Exo48, Exo49, Exo50, Exo51
, Exo52, Exo53, Exo54, Exo55, Exo56, Exo57
, Exo58, Exo59, Exo60, Exo61, Exo62, Exo63
, Exo64, Exo65, Exo66, Exo67, Exo68, Exo69
, Exo70, Exo71, Exo72, Exo73, Exo74, Exo75
, Exo76, Exo77, Exo78, Exo79, Exo80, Exo81
, Exo82, Exo83, Exo84, Exo85, Exo86, Exo87
, Exo88, Exo89, Exo90, Exo91, Exo92, Exo93
, Exo94, Exo95, Exo96, Exo97, Exo98, Exo99
, Exo100, Exo101, Exo102, Exo103, Exo104,
Exo105, Exo106, Exo107, Exo108, Exo109, E
xo110, Exo111, Exo112, Exo113, Exo114, Ex
Nucleic acids encoding o115, Exo116, Exo117, and Exo118, as well as these Exo proteins, are provided.

In a further aspect, the invention provides a method of producing an Exo protein, the method comprising providing a cell comprising an Exo nucleic acid and subjecting the cell to conditions that allow expression of the Exo protein. Including putting.

In a further aspect, the invention provides a method of screening for bioactive agents capable of binding to Exo protein. The method consists of binding the Exo protein to a candidate bioactive agent and measuring the binding of the candidate bioactive agent to the Exo protein.

In another aspect, the invention provides a method of screening for agents capable of interfering with the binding of Exo protein to GS27. This method is
binding the o protein, the candidate bioactive agent and the GS27 protein, and measuring the binding of the Exo protein to the GS27 protein.

In another aspect, the invention provides a method of screening for agents capable of interfering with the binding of Exo protein to rab7. The method involves binding Exo protein, a candidate bioactive agent and rab7 protein, and measuring binding of Exo protein to rab7 protein.

In a further aspect, the present invention provides a method of screening for agents capable of interfering with the binding of rab9 to Exo protein. This method is
Binding the xo protein, the candidate bioactive agent and the rab9 protein, and measuring the binding of the Exo protein to the rab9 protein.

In yet another aspect, the invention features an Exo protein and a snap.
Methods of screening for agents capable of interfering with -23 binding are provided. This method involves Exo protein, candidate bioactive agents and snap-23.
Bind protein and measure binding of Exo protein to snap-23 protein.

In an additional aspect, the invention provides a method of screening for agents capable of interfering with Exo protein and rab3a binding. The method involves binding the Exo protein, a candidate bioactive agent and the rab3a protein, and measuring the binding of the Exo protein and the rab3a protein.

In another aspect, the invention provides a method of screening for agents capable of interfering with Exo protein and rab11 binding. The method involves binding the Exo protein, a candidate bioactive agent and the rab11 protein, and measuring the binding of the Exo protein and the rab11 protein.

In a further aspect, the invention provides a method of screening for agents capable of interfering with the binding of Exo protein and rab3d. The method involves binding the Exo protein, a candidate bioactive agent and the rab3d protein, and measuring the binding of the Exo protein and the rab3d protein.

In yet an additional aspect, the invention provides a method of screening for agents capable of interfering with Exo protein and rab5 binding. The method involves binding Exo protein, a candidate bioactive agent and rab5 protein, and measuring the binding of Exo protein and rab5 protein.

In another aspect, the invention features an Exo protein and an alpha-s
Methods of screening for agents capable of interfering with the binding of nap are provided. This method involves Exo protein, candidate bioactive agents and alpha-s
binding to nap protein, and Exo protein and alpha
measuring the binding of a-snap protein.

In yet another embodiment, the invention provides an Exo protein and unc18
A method of screening for agents capable of interfering with -1 binding is provided. The method involves binding Exo protein, a candidate bioactive agent and unc18-1 protein, and measuring binding of Exo protein and unc18-1 protein.

In another aspect, the invention provides a method of screening for agents capable of interfering with Exo protein and vamp3 binding. The method involves binding Exo protein, a candidate bioactive agent and vamp3 protein, and measuring binding of Exo protein and vamp3 protein.

In another aspect, the invention provides a method of screening for bioactive agents capable of modulating the activity of Exo proteins. This method is
adding a candidate bioactive agent to cells consisting of a recombinant nucleic acid encoding an o protein and measuring the effect of the candidate bioactive agent on cellular activity. In a preferred embodiment, the cell activity is exocytosis or vesicle trafficking.

In another aspect, the invention provides a method of treating a disease associated with exocytosis, which comprises a protein selected from those shown in the sequence listing, such that the disease is ameliorated. GS27, Rab3a, expressed in tissues,
Rab7, Rab9, Rab11, Rab3d, Rab5, alpha-sna
administering an agent that interferes with specific binding to a protein selected from the group consisting of p, unc18-1, vamp3 and snap-23.

Also provided herein is an agent that binds to a patient an agent that binds to a protein encoded by a sequence selected from the group consisting of those listed in the sequence listing so that exocytosis is altered. A method of treating a disease associated with exocytosis, comprising administering.

Further provided herein is a method of reducing or inhibiting exocytosis in a cell, which is SEQ ID NOS: 1-51 (odd) and 53.
A protein selected from those encoded by a sequence selected from the group consisting of -211 and GS27, Rab3a, Rab7, Rab9, Rab11, expressed in the cell such that exocytosis is inhibited, Rab3d, Ra
b5, alpha-snap, unc18-1, vamp3 and snap-2.
Administering an agent that interferes with specific binding to a protein selected from the group consisting of 3.

In yet another aspect, the present invention provides SEQ ID NOS: 1-51 (
(Odd number) and 53-211, which provides a method of neutralizing the effect of a protein encoded by a sequence, which is sufficient to effect neutralizing agents specific for the protein. Contacting with a different amount of the protein. Other aspects of the invention are described as described below.

BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A and 1B show the basic mechanism of the yeast two-hybrid and yeast one-hybrid systems. Figure 1A shows a yeast two-hybrid: GAL4A is G
Represents the AL4 transcriptional activation domain. cDNA represents the cDNA library insert. X represents any decoy gene. GAL4B represents the GAL4 DNA binding domain. HIS / lacZ has a reporter gene of HIS or l
It is either acZ.

FIG. 2 shows a schematic of the yeast two-hybrid screen. Closed black dots represent colonies on the plate. Transformation steps for both the bait plasmid and the cDNA library plasmid are shown.

FIG. 3 shows a schematic of the yeast one-hybrid screen. Closed black dots represent colonies on the plate.

4A-4D show the vectors used in the yeast two-hybrid and yeast one-hybrid screens, respectively. 4A-4B show a two-hybrid vector. The decoy vector is pHyflex / Zeo (Invitro
gen), pBD-GAL4 (Stratagene), pAS2-1 (Clo).
nTech) or pGilda (Origene, Clontech)
May be The arrow indicates the transformation of the fusion protein on either the bait or the cDNA vector. The binding domain is GAL4 or LexA
Either can be used. Underlined MCS represent multiple cloning sites, where either the bait gene or the cDNA fragment must be cloned. 2 μOri represents the yeast 2 micron origin of replication. The cDNA vector is pYE
STrp2 (Invitrogen), pAD-GAL4 (Stratagen)
e), pACT2 (Clontech), pGADGH (Clontech)
, PGAD424 (Clontech) or pJG4-5 (Origene)
Company). The activation domain may be GAL4, VP16 or other transforming activator. 4C-4D show a one-hybrid reporter vector. The DNA sequence of interest is an underlined multiple cloning site (
Must be inserted in the MCS). The enzyme used to linearize the reporter vector for integration is indicated by the solid arrow. The dotted arrow is HIS
Alternatively, transformation of either the lacZ gene is shown.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides Exo proteins and nucleic acids involved in the exocytosis pathway. In a preferred embodiment, the Exo protein is of vertebrate origin,
And more preferably, rodents (rat, mouse, hamster, guinea pig, etc.)
, Mammals, including primates, farm animals (including sheep, goats, pigs, cows, horses, etc.), and in a most preferred embodiment, humans.

The Exo proteins of the present invention can be identified in several ways. “Protein” in this sense includes proteins, polypeptides and peptides. Ex of this invention
o proteins fall into two general classes: they may have homology to either known proteins or expressed sequence tags (ESTs), but at the time of discovery they are completely new to public databases. protein. Alternatively, known proteins, but not known to be involved in exocytosis; ie, they are Exo proteins identified herein as having novel biological functions. Thus, Exo proteins may initially be identified as being related to proteins known to be involved in exocytosis or vesicle trafficking. In one embodiment provided by the present invention, the Exo protein is GS2.
7, rab7, rab9, snap-23, rab3a, rab11, rab3
It binds to a protein selected from the group consisting of d, rab5, alpha-snap, unc18-1 and vamp3. Exo proteins may be new or known to exist in the art, including GS27, rab7,
lab9, snap-23, lab3a, lab11, lab3d, lab5,
It is not known to bind to alpha-snap, unc18-1 and vamp3. When these Exo proteins and nucleic acids are novel, their compositions and methods of use are provided herein. These Exo proteins and nucleic acids are known but GS27, rab7, rab9, snap-23
, Lab3a, lab11, lab3d, lab5, alpha-snap, u
If not known to bind nc18-1 and vamp3, a method of their use, ie functional screening, is provided.

In one embodiment, the Exo nucleic acid or Exo protein is first identified by substantial nucleic acid and / or amino acid sequence identity or similarity to the sequences provided herein. In a preferred embodiment, the Exo nucleic acid or Exo protein has sequence identity or similarity to the sequences provided herein below and binds to an exocytosis or vesicle transport protein. Preferred exocytosis and vesicle transport proteins are GS27, rab7, rab9, snap-23, rab3a, rab.
11, rab3d, rab5, alpha-snap, unc18-1 and v
including amp3 (these proteins known in the art are considered identical, even if a "-" is used in the name or capital letters are used). The identity or similarity of such sequences can be based on the overall nucleic acid or amino acid sequence.

SEQ ID NO: 1 is described in Hay JC, et al., J Cell Biol 1998, 141 (7): 1489-1502. Mouse syntaxin 4, Genbank access no. : U76832, showing a nucleic acid sequence that encodes at least a portion of:

SEQ ID NO: 3 is Burbelo PD, et al. Gene 1994, 139 (2): 241-245.
LZIP-1 and LZIP-2, Genbank Access no. : AC003675, showing a nucleic acid sequence encoding at least part of

SEQ ID NO: 5 is Tabira T, et al., Ann NY Acad Sci. 1998, 840: 10.
7-116., Mouse IL-3 receptor, Genbank access no. :
3 shows a nucleic acid sequence encoding at least a portion of M29855.

SEQ ID NO: 7 is Ryan JJ, et al., J Immunol. 1998, 161 (4): 181.
1-1821., Mouse IL-4 receptor, access no. : M27959
, A nucleic acid sequence encoding at least part of

SEQ ID NO: 9 is Ryan JJ, et al., J Immunol. 1998, 161 (4): 181.
1-1821., Mouse IL-4 receptor, access no. : M27959
A second nucleic acid sequence encoding at least a portion of

SEQ ID NO: 11 is Brown, SD, et al., Biochem. Biophys. Res. C
ommun. 248 (3): 879-888 (1998), mouse LDL receptor-related protein 6 (Lrp6), access no. : AF074265, showing a nucleic acid sequence encoding at least a part thereof.

SEQ ID NO: 13 is from Illing M, et al., J Biol Chem 1997, 11: 272 (
15): 10303-10310, mouse abc2, access no. : X7592
7 shows a nucleic acid sequence encoding at least a part of 7.

SEQ ID NOS: 15, 17, 19, 21, 23, and 25 represent nucleic acid sequences encoding Exo3-8, respectively.

SEQ ID NO: 27 is Hay JC, et al., J Cell Biol. 1998, 141 (7): 148.
9-1502, Exo9, which may share some features with human syntaxin 16A, access no. Shows a nucleic acid sequence encoding: AF008937.

SEQ ID NO: 29 is a putative RNA-binding protein (RBP56) described in Genomics 38: 51-57 (1996), access no. : U51334, which shows a nucleic acid sequence encoding Exo10 that may share some features.

SEQ ID NO: 31 is GenBank access no. : AA14408
3 shows a nucleic acid sequence encoding Exo11 with some similarity to 3.

SEQ ID NO: 33 is GenBank access no. : AA10318
5 shows a nucleic acid sequence encoding Exo12 with some similarity to 5.

SEQ ID NO: 35 is GenBank access no. : AA91922
2 shows a nucleic acid sequence encoding Exo13 with some similarity to 2.

SEQ ID NO: 37 is GenBank access no. : AA27601
5 and nucleic acid sequences encoding Exo14 with some similarity to human (xs99).

SEQ ID NO: 39 is GenBank access no. : AA61726
6 and CREB-RP (creb-rp), GenBank access no. : U
31903 shows a nucleic acid sequence encoding Exo15 with some similarities to 31903.

SEQ ID NO: 41 is GenBank access no. : AA22129
3 shows nucleic acid sequences encoding Exo16 with some similarity to 3 and rat plate related peptides.

SEQ ID NO: 43 is Rowe T, et al., Science 1998 279 (5351): 696-7.
GenBank access no. : AA166109 and rat syntaxin 5, Genbank Access no. : Shows a nucleic acid sequence encoding Exo17a with some similarity to L20822.

SEQ ID NO: 45 is Rowe T, et al., Science 1998 279 (5351): 696-7.
GenBank access no. : AA166109 and rat syntaxin 5, Genbank Access no. : Shows a nucleic acid sequence encoding Exo17b having some similarity to L20822.

SEQ ID NO: 47 is Rowe T, et al., Science 1998 279 (5351): 696-7.
Genbank access no. : AA166109 with some similarities and rat syntaxin 5, GenBank Access no. :
3 shows the nucleic acid sequence encoding Exo18 with some similarity to L20822.

SEQ ID NO: 49 is described in Hay JC, J Cell Biol 1998 141 (7): 1489-1502., GenBank Access No. : Shows nucleic acid sequences encoding Exo19 with some similarity to U76832 and mouse syntaxin 4.

[0073]   SEQ ID NO: 51 shows a nucleic acid sequence encoding Exo20.

[0074]   SEQ ID NO: 53 shows a nucleic acid sequence encoding Exo21.

SEQ ID NO: 54 is a human synaptic vesicle axon transporter, Genbank Access no. : Shows a nucleic acid sequence encoding a portion of X90840.

SEQ ID NO: 55 is the human cargo selection protein TIP47 (TI
P47), Genbank access no. : Shows a nucleic acid sequence encoding a portion of AF057140.

SEQ ID NO: 56 is the human cargo selection protein TIP47 (TI
P47), Genbank access no. : Shows a nucleic acid sequence encoding a portion of AF057140.

SEQ ID NO: 57 is the human cargo selection protein TIP47 (TI
P47), Genbank access no. : Shows a nucleic acid sequence encoding a portion of AF057140.

SEQ ID NO: 58 is the human cargo selection protein TIP47 (TI
P47), Genbank access no. : Shows a nucleic acid sequence encoding a portion of AF057140.

SEQ ID NO: 59 is a human tax interacting protein, Genbank
Access no. : Shows a nucleic acid sequence encoding a portion of AF028882.

SEQ ID NO: 60 is a human tax interacting protein, Genbank
Access no. : Shows a nucleic acid sequence encoding a portion of AF028882.

SEQ ID NO: 61 is a human tax interacting protein, Genbank
Access no. : Shows a nucleic acid sequence encoding a portion of AF028882.

SEQ ID NO: 62 is human human inositol polyphosphate 5-phosphatase, Genbank Access no. : Shows a nucleic acid sequence encoding a portion of M74161.

[0084]   SEQ ID NO: 63 shows a nucleic acid sequence encoding Exo22.

[0085]   SEQ ID NO: 64 shows a nucleic acid sequence encoding Exo23.

SEQ ID NO: 65 is a mouse C57BL / 6JSec61 protein complex gamma subunit; Genbank Access no. : Shows a nucleic acid sequence encoding a portion of U11027.

SEQ ID NO: 66 is mouse HMG-1; GenBank access no
． : Shows a nucleic acid sequence encoding a portion of U00431.

SEQ ID NO: 67 is mouse cyclin B2; GenBank access no. : Shows a nucleic acid sequence encoding a portion of X66032.

SEQ ID NO: 68 is mouse cyclin B2; GenBank access no. : Shows a nucleic acid sequence encoding a portion of X66032.

SEQ ID NO: 69 is mouse pancreatic beta cell kinesin heavy chain; GenBan
k access no. : Shows a nucleic acid sequence encoding a portion of U86090.

SEQ ID NO: 70 is a mouse pancreatic beta cell kinesin heavy chain; GenBan
k access no. : Shows a nucleic acid sequence encoding a portion of U86090.

SEQ ID NO: 71 is mouse syntaxin 4; GenBank Access no. : Shows a nucleic acid sequence encoding a portion of U76832.

SEQ ID NO: 72 is mouse syntaxin 4; GenBank Access no. : Shows a nucleic acid sequence encoding a portion of U76832.

SEQ ID NO: 73 is mouse syntaxin 4; GenBank Access no. : Shows a nucleic acid sequence encoding a portion of U76832.

SEQ ID NO: 74 is mouse stearoyl-CoA desaturase (S
CD2); GenBank access no. : Shows a nucleic acid sequence encoding a portion of M26270.

SEQ ID NO: 75 is mouse spermidine aminopropyl transferase (Mspmsy); GenBank Access no. : Shows a nucleic acid sequence encoding a portion of AF031486.

SEQ ID NO: 76 is mouse prothymosin alpha; GenBank Access no. : Shows a nucleic acid sequence encoding a portion of X56135.

SEQ ID NO: 77 is a mouse protein cofactor; GenBank
Access no. : Shows a nucleic acid sequence encoding a portion of U74079.

SEQ ID NO: 78 is mouse external defense fiber protein 2 (Odf2); G
enBank access no. : Shows a nucleic acid sequence encoding a portion of AF000968.

SEQ ID NO: 79 is a mouse protein expressed in E12 brain (clone C2); GenBank Access no. : Shows a nucleic acid sequence encoding a portion of X83589.

SEQ ID NO: 80 is a mouse hnRNP K homolog; GenBan
k access no. : Shows a nucleic acid sequence encoding a portion of L29769.

SEQ ID NO: 81 is mouse NRF1 (NFE2-related factor 1); Gen
Bank access no. : Shows a nucleic acid sequence encoding a portion of X78709.

SEQ ID NO: 82 is a mouse RNA binding protein; GenBank Access no. : Shows a nucleic acid sequence encoding a portion of L17076.

SEQ ID NO: 83 is mouse Dynactin 1; GenBank Access no. : Shows a nucleic acid sequence encoding a portion of U60312.

SEQ ID NO: 84 is a mouse hormone sensitive lipase; GenBank
Access no. : Shows a nucleic acid sequence encoding a portion of U08188.

SEQ ID NO: 85 is a mouse mtprda (human TPRD homolog); G
enBank access no. : Shows a nucleic acid sequence encoding a portion of AB008516.

SEQ ID NO: 86 shows the nucleic acid sequence encoding Exo24, Ge
nBank access no. : Has some similarity to AA2685651.

SEQ ID NO: 87 shows the nucleic acid sequence encoding Exo25, Ge
nBank access no. : Has some similarity to AA097037.

SEQ ID NO: 88 shows a nucleic acid sequence encoding Exo26, Ge
nBank access no. : Has some similarity to AA097037.

SEQ ID NO: 89 shows the nucleic acid sequence encoding Exo27, Ge
nBank access no. : Has some similarity to AA259474.

SEQ ID NO: 90 represents a nucleic acid sequence encoding Exo28, Ge
nBank access no. : Has some similarity to AA555886.

SEQ ID NO: 91 shows the nucleic acid sequence encoding Exo29, Ge
nBank access no. : Has some similarity to AA770839.

SEQ ID NO: 92 shows the nucleic acid sequence encoding Exo30, Ge
nBank access no. : Has some similarity to AA770839.

SEQ ID NO: 93 shows the nucleic acid sequence encoding Exo31, Ge
nBank access no. : Has some similarity to AA415504.

SEQ ID NO: 94 shows the nucleic acid sequence encoding Exo32, Ge
nBank access no. : Has some similarity to AA415504.

SEQ ID NO: 95 shows the nucleic acid sequence encoding Exo33, Ge
nBank access no. : Has some similarity to AA415504.

SEQ ID NO: 96 shows the nucleic acid sequence encoding Exo34, Ge
nBank access no. : Has some similarity to AA415504.

SEQ ID NO: 97 shows the nucleic acid sequence encoding Exo35, Ge
nBank access no. : Has some similarity to AA415504.

SEQ ID NO: 98 shows the nucleic acid sequence encoding Exo36, Ge
nBank access no. : Has some similarity to AA415504.

SEQ ID NO: 99 shows the nucleic acid sequence encoding Exo37, Ge
nBank access no. : Has some similarity to AA415504.

SEQ ID NO: 100 shows the nucleic acid sequence encoding Exo38, G
enBank access no. : Has some similarity to AA415504.

SEQ ID NO: 101 shows the nucleic acid sequence encoding Exo39, G
enBank access no. : Has some similarity to AA415504.

SEQ ID NO: 102 represents a nucleic acid sequence encoding Exo40, G
enBank access no. : Has some similarity to AA415504.

SEQ ID NO: 103 shows the nucleic acid sequence encoding Exo41, G
enBank access no. : Has some similarity to AA172925.

SEQ ID NO: 104 shows a nucleic acid sequence encoding Exo42, G
enBank access no. : Has some similarity to AA288130.

SEQ ID NO: 105 shows the nucleic acid sequence encoding Exo43, G
enBank access no. : Has some similarity to AI181639.

SEQ ID NO: 106 shows the nucleic acid sequence encoding Exo44, G
enBank access no. : Has some similarity to AA184709.

SEQ ID NO: 107 shows the nucleic acid sequence encoding Exo45, G
enBank access no. : Has some similarity to AA266406.

SEQ ID NO: 108 shows a nucleic acid sequence encoding Exo46, G
enBank access no. : Has some similarity to AA563185.

SEQ ID NO: 109 shows a nucleic acid sequence encoding Exo47, G
enBank access no. : Has some similarity to AA519170.

SEQ ID NO: 110 shows the nucleic acid sequence encoding Exo48, G
enBank access no. : Has some similarity to AA519170.

SEQ ID NO: 111 represents a nucleic acid sequence encoding Exo49, yeast ORMI, GenBank Access no. : Has some similarity to AA175198.

SEQ ID NO: 112 shows a nucleic acid sequence encoding Exo50, which is obtained from rat mt-GrpE no. 1 precursor, GenBank Access no. : AA0
It has some similarities to 60861.

SEQ ID NO: 113 shows a nucleic acid sequence encoding Exo51, human CENP-F centromere protein, GenBank Access no. : AI034
It has some similarities with 171.

SEQ ID NO: 114 represents a nucleic acid sequence encoding Exo52, human arfaptin2 (putative target of ADP-ribosylation factor), GenBank
Access no. : Has some similarity to AA543955.

SEQ ID NO: 115 represents a nucleic acid sequence encoding Exo53, human brain and regenerating organ-expressed protein (BRE); GenBank Access no.
: Has some similarity to AA200608.

SEQ ID NO: 116 represents a nucleic acid sequence encoding Exo54, human brain and regenerating organ-expressed protein (BRE); GenBank Access no.
: Has some similarity to AA200608.

SEQ ID NO: 117 shows the nucleic acid sequence encoding Exo55, human cell cycle promoting 2 protein (CPR2); GenBank Access no. : WB
It has some similarities to 87077.

SEQ ID NO: 118 shows a nucleic acid sequence encoding Exo56, which is a spliceosome associated protein (SAP145); GenBank Access no.
: Has some similarity to Al119401.

SEQ ID NO: 119 shows the nucleic acid sequence encoding Exo57, m
esocricetus auratus stearyl-CoA desaturase
(FAR-17c); GenBank access no. : Has some similarity to AA387696.

SEQ ID NO: 120 represents a nucleic acid sequence encoding Exo58, rat inositol triphosphate receptor subtype 3 (IP3R-3); GenBan
k access no. : Has some similarity to AA823026.

SEQ ID NO: 121 shows the nucleic acid sequence encoding Exo59, L
-Asparaginase; GenBank Access no. : Has some similarity to AI118730.

SEQ ID NO: 122 shows the nucleic acid sequence encoding Exo60, L
-Asparaginase; GenBank Access no. : Has some similarity to Al118730.

SEQ ID NO: 123 represents a nucleic acid sequence encoding Exo61, human RB-binding protein (RBBP-2); GenBank Access no. : AA
It has some similarities to 755315.

SEQ ID NO: 124 represents a nucleic acid sequence encoding Exo62, human secretory apoptotic protein 3 (SARP3); GenBank Access no. : Has some similarity to AU018890.

SEQ ID NO: 125 represents a nucleic acid sequence encoding Exo63, a myosin heavy chain; GenBank Access no. : Has some similarity to AA237764.

SEQ ID NO: 126 represents a nucleic acid sequence encoding Exo64, a myosin heavy chain; GenBank Access no. : Has some similarity to AA237764.

SEQ ID NO: 127 shows the nucleic acid sequence encoding Exo65, myosin heavy chain; GenBank Access no. : Has some similarity to AA237764.

SEQ ID NO: 128 represents a nucleic acid sequence encoding Exo66, a myosin heavy chain; GenBank Access no. : Has some similarity to AA237764.

SEQ ID NO: 129 represents a nucleic acid sequence encoding Exo67, rat tomosin; GenBank Access no. : Has some similarity to AA437465.

SEQ ID NO: 130 shows a nucleic acid sequence encoding Exo68, rat tomosin; GenBank Access no. : Has some similarity to AA437465.

SEQ ID NO1: 31 represents a nucleic acid sequence encoding Exo69, rat tomosin; GenBank Access no. : Has some similarity to AA437465.

SEQ ID NO: 132 represents a nucleic acid sequence encoding Exo70, rat tomosin; GenBank Access no. : Has some similarity to AA437465.

SEQ ID NO: 133 shows the nucleic acid sequence encoding Exo71, human mcag29CTG repeat region; GenBank Access no. : AA0893
It has some similarities with 40.

SEQ ID NO: 134 represents a nucleic acid sequence encoding Exo72, a rat G protein gamma-5 subunit; GenBank Access no. : A
It has some similarities to A021879.

SEQ ID NO: 135 represents a nucleic acid sequence encoding Exo73, a rat G protein gamma-5 subunit; GenBank Access no. : A
It has some similarities to A021879.

SEQ ID NO: 136 shows the nucleic acid sequence encoding Exo74, rat G protein gamma-5 subunit; GenBank Access no. : A
It has some similarities to A021879.

SEQ ID NO: 137 represents a nucleic acid sequence encoding Exo75, rat G protein gamma-5 subunit; GenBank Access no. : A
It has some similarities to A021879.

SEQ ID NO: 138 represents a nucleic acid sequence encoding Exo76, a rat G protein gamma-5 subunit; GenBank Access no. : A
It has some similarities to A021879.

SEQ ID NO: 139 represents a nucleic acid sequence encoding Exo77, rat G protein gamma-5 subunit; GenBank Access no. : A
It has some similarities to A021879.

[0161]   SEQ ID NO: 140 shows a nucleic acid sequence encoding Exo78.

[0162]   SEQ ID NO: 141 shows a nucleic acid sequence encoding Exo79.

[0163]   SEQ ID NO: 142 shows a nucleic acid sequence encoding Exo80.

[0164]   SEQ ID NO: 143 shows the nucleic acid sequence encoding Exo81.

SEQ ID NO: 144 is human HLA-B related transcript 3; GenBan
k access no. : Shows a nucleic acid sequence encoding a portion of M33519.

SEQ ID NO: 145 is human HLA-B related transcript 3; GenBan
k access no. : Shows a nucleic acid sequence encoding a portion of M33519.

SEQ ID NO: 146 for Human T cell leukemia / lymphoma 1; GenBan
k access no. : Shows a nucleic acid sequence encoding a portion of X82240.

[0168]   SEQ ID NO: 147 shows a nucleic acid sequence that encodes Exo82.

SEQ ID NO: 148 represents a nucleic acid sequence encoding Exo83; likely to have some homology with rat rabin3.

SEQ ID NO: 149 is at least human KIAA0665; GenBa
nk access no. : Shows a nucleic acid sequence encoding a portion of AB014565.

SEQ ID NO: 150 is Rabin 3; GenBank access no.
: Shows a nucleic acid sequence encoding Exo84 that may have some similarities to AA846576.

SEQ ID NO: 151 is Rabin 3; GenBank access no.
: Shows a nucleic acid sequence encoding Exo85 that may have some similarity to AA846576.

SEQ ID NO: 152 is KIAA0665; GenBank Access n
o. : Shows a nucleic acid sequence encoding Exo86 that may have some similarity to AA757034.

SEQ ID NO: 153 is a mouse protein cofactor; GenBan
k access no. : Shows a nucleic acid sequence encoding a portion of U74079.

SEQ ID NO: 154 is a mouse protein cofactor; GenBan
k access no. : Shows a nucleic acid sequence encoding a portion of U74079.

SEQ ID NO: 155 represents a nucleic acid sequence encoding Exo87, a calcium-dependent protein kinase, Genbank Access no. : AA7
Will have some similarities to 70736.

SEQ ID NO: 156 represents a nucleic acid sequence encoding Exo88, a calcium-dependent protein kinase, Genbank Access no. : AA7
Will have some similarities to 70736.

SEQ ID NO: 157 represents a nucleic acid sequence encoding Exo89, a calcium-dependent protein kinase, Genbank Access no. : AA7
Will have some similarities to 70736.

SEQ ID NO: 158 represents a nucleic acid sequence encoding Exo90, a calcium-dependent protein kinase, Genbank Access no. : AA7
Will have some similarities to 70736.

SEQ ID NO: 159 represents a nucleic acid sequence encoding Exo91, human mcag29 CTG repeat region, Genbank Access no. : AA473
325 will have some similarities.

SEQ ID NO: 160 represents a nucleic acid sequence encoding Exo92, human mcag29 CTG repeat region, Genbank Access no. : AA473
325 will have some similarities.

SEQ ID NO: 161 shows the nucleic acid sequence encoding Exo93, G
enbank access no. : Will have some similarities to AA138122.

SEQ ID NO: 162 shows the nucleic acid sequence encoding Exo94, G
enbank access no. : Will have some similarities to AA138122.

SEQ ID NO: 163 shows the nucleic acid sequence encoding Exo95, G
enbank access no. : Will have some similarities to AA138122.

SEQ ID NO: 164 shows the nucleic acid sequence encoding Exo96, G
enbank access no. : Will have some similarities to AA138122.

SEQ ID NO: 165 shows the nucleic acid sequence encoding Exo97, G
enbank access no. : Will have some similarities to AA138122.

SEQ ID NO: 166 shows the nucleic acid sequence encoding Exo98, G
enbank access no. : Will have some similarities to AA060976.

SEQ ID NO: 167 shows the nucleic acid sequence encoding Exo99, G
enbank access no. : Will have some similarities to AA277208.

SEQ ID NO: 168 represents a nucleic acid sequence encoding Exo100,
Genbank access no. : Will have some similarities to AA277208.

SEQ ID NO: 169 shows the nucleic acid sequence encoding Exo101,
Genbank access no. : Will have some similarities to AA467477.

SEQ ID NO: 170 shows the nucleic acid sequence encoding Exo102,
Genbank access no. : Will have some similarities to AA467477.

SEQ ID NO: 171 shows the nucleic acid sequence encoding Exo103,
Genbank access no. : Will have some similarities to AA833213.

SEQ ID NO: 172 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 173 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 174 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 175 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 176 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 177 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 178 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 179 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 180 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 181 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 182 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 183 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 184 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 185 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 186 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 187 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 188 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 189 is Rab2; GenBank access no. : X
9 shows a nucleic acid sequence encoding a portion of 95403.

SEQ ID NO: 190 is Rab5c; GenBank access no. :
3 shows a nucleic acid sequence encoding a portion of AA230407.

SEQ ID NO: 191 is Rab5c; GenBank access no. :
3 shows a nucleic acid sequence encoding a portion of AA230407.

SEQ ID NO: 192 is Rab5c; GenBank access no. :
3 shows a nucleic acid sequence encoding a portion of AA230407.

[0214]   SEQ ID NO: 193 shows a nucleic acid sequence encoding Exo104.

SEQ ID NO: 194 shows a nucleic acid sequence encoding a human gene, Exo105, which is similar to the mouse testis-specific protein PBS13, and is described in GenBank Access no. : Will have some similarities to AA184365.

SEQ ID NO: 195 shows the nucleic acid sequence encoding Exo106,
GenBank access no. : Will have some similarities to AA504490.

SEQ ID NO: 196 shows the nucleic acid sequence encoding Exo107,
GenBank access no. : Will have some similarities to AA504490.

SEQ ID NO: 197 shows the nucleic acid sequence encoding Exo108,
GenBank access no. : Will have some similarities to AI181750.

SEQ ID NO: 198 shows the nucleic acid sequence encoding Exo109,
GenBank access no. : Will have some similarities to AI181750.

SEQ ID NO: 199 shows the nucleic acid sequence encoding Exo110,
GenBank access no. : Will have some similarities to AU043111.

SEQ ID NO: 200 shows the nucleic acid sequence encoding Exo111, a human gene similar to rat alpha-soluble NSF adhesion protein (SNAP).

SEQ ID NO: 201 shows the nucleic acid sequence encoding Exo112,
3 shows a nucleic acid sequence encoding a human gene, Exo105, similar to the mouse testis-specific protein PBS13; GenBank Access no. : AA184365
Would have some similarities to.

SEQ ID NO: 202 shows the nucleic acid sequence encoding Exo113 which appears to be similar to the mouse zinc finger protein.

SEQ ID NO: 203 shows the nucleic acid sequence encoding Exo114 which appears to be similar to the mouse zinc finger protein.

SEQ ID NO: 204 shows a nucleic acid sequence encoding Exo115 which appears to resemble chicken c-hairy1; GenBank Access no.
: Will have some similarities to AA116067.

SEQ ID NO: 205 shows a nucleic acid sequence encoding Exo116 that appears to resemble chicken c-hairy1; GenBank Access no.
: Will have some similarities to AA116067.

SEQ ID NO: 206 is mouse syntaxin 4; GenBank Access no. : Shows a nucleic acid sequence encoding a portion of U76832.

SEQ ID NO: 207 is a mouse interleukin (IL) -3 receptor; G
enBank access no. : Shows a nucleic acid sequence encoding a portion of M29855.

SEQ ID NO: 208 is mouse interleukin (IL) -3 receptor; G
enBank access no. : Shows a nucleic acid sequence encoding a portion of M29855.

SEQ ID NO: 209 is a mouse low density lipoprotein (LDL) receptor-
Related proteins; GenBank Access no. : Shows a nucleic acid sequence encoding a portion of AF074265.

SEQ ID NO: 210 is similar to human ANF126 zinc protein,
1 shows the nucleic acid sequence encoding Exo117.

SEQ ID NO: 211 shows a nucleic acid sequence encoding Exo118, which is similar to the rat isoprenylated 67 kDa protein.

As indicated above, Exo3 to Exo118 are new. SEQ ID N
Exo proteins encoded by OS: 1-51 (odd number) and sequence ID NOS: 53-211 are shown for the first time in the present invention to bind them to exocytosis or vesicle transport proteins or fragments thereof. That is, each is new. In a preferred embodiment, SEQ ID N
OS: Proteins encoded by 1-51 (odd number) bind to GS27; SE
The protein encoded by Q ID NOS: 53 binds to rab7; S
The protein encoded by EQ ID NOS: 54-64 binds to rab9; the protein encoded by SEQ ID NOS: 65-143 binds to snap-23; encoded by SEQ ID NOS: 144-148 Protein binds to rab3a; SEQ ID NOS: 149-152
Is encoded by rab11; SEQ ID NOS:
The protein encoded by 153-171 binds to rab3d; SEQ
The proteins encoded by ID NOS: 172-193 bind to rab5; the proteins encoded by SEQ ID NOS: 194-201 bind to alpha-snap; the proteins encoded by SEQ ID NOS: 202-205 Binds to unc18-1; and SEQ ID NOS
The proteins encoded by: 206-211 bind to vamp3.

In a preferred embodiment, if SEQ ID NOS: 1-51, odd number,
And SEQ ID NOS: 53-211, preferably a sequence encoding Exo3-118, having a total sequence identity of any one of the protein sequences with any one of the amino acid sequences preferably greater than or equal to about 75%, More preferably 80% or more, even more preferably 85% or more, and most preferably 90% or more, the protein is an "Exo protein". In some embodiments, the sequence identity will be as high as about 93 to 95 or 98%. As is known in the art, a number of different programs can be used to determine whether they have sequence identity or similarity to a known gene or expressed sequence tag (EST). Sequence identity is measured using, but is not limited to, the following standard techniques known in the art: Smith & Waterman, Adv.
ppl. Math. 2: 482 (1981) Local Sequence Confirmation Algorithm, Needdleman & Wunsch,
J. Mol. Biool. 48: 443 (1970) Sequence Confirmation Alignment Algorithm, Pearson
& Lipman, PNAS USA 85: 2444 (1988) similarity search method, these algorithms (W
isconsin Genetics Software Package, Genetics Computer Group, 575 Science
GAP, BESTFIT, FASTA, and T at Drive, Madison, WI
FASTA) computer implementation, Devereux et al., Nucl. Acid Res.
12: 387-395 (1984), The Best Fit Sequence Program, preferably by default or by inspection. Preferably the percent identity is FastD
Calculated by B based on the following parameters: Mismatch penalty 1
Gap Penalty 1; Gap Size Penalty 0.33; and Penalty Penalty 30, "Current Methods in Sequence Comparison and Analysis,"
Macromolecule Sequencing and Synthesis, Selected Methods and Application
s, PP 127-149 (1988), Alan R. Liss, Inc.

One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using a progressive, pairwise alignment. It can also draw trees that show clustering relationships and create their alignment. PILEUP is Feng & Doolittle, J. Mol. Evol. 35: 351-36
0 (1987) using the simplification of the forward alignment method; the method is Higgins & S
Similar to that described in harp CABIOS 5: 151-153 (1989). Useful PILEUP parameters are default gap load 3.00, default gap length load 0.1
0, and loaded end gap.

Examples of other useful algorithms are Altschul et al., J. Mol. Biol. 215, 403-4.
10, (1990) and Karlin et al., PNAS USA 90: 5837-5787 (1993), B.
The LAST algorithm. A particularly useful BLAST program is Altschul et
al., Methods in Enzymology, 266: 460-480 (1996); http: //blast.wustl/edu/
blast / README.html] is a WU-BLAST-2 program. W
U-BLAST-2 uses several search parameters, most of which are set to default values. The adjustable parameters are set to the following values: overlap range = 1, overlap ratio = 0.125, word threshold (T) = 11. HSPS and HS
The PS2 parameter is a dynamic value and is established by the program itself according to the particular sequence composition and the particular database composition from which the sequence of interest is searched; however, its value will be adjusted to increase sensitivity. . % Amino acid sequence identity is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
The "longer" sequence is the one with the most practical residues in the aligned regions (ignoring the gaps introduced by WU-BLAST-2 to maximize the alignment score).

Similarly, the “percentage (%) nucleic acid sequence identity to a coding sequence of a polypeptide identified herein is identical to the nucleotide residue in the coding sequence of the Exo protein of a nucleotide residue in a candidate sequence. Defined as a percentage, one preferred method is WU-BLAST-set to default parameters.
Utilizing 2 BLASTN modules, set the overlap range and overlap ratio to 1 and 0.125 respectively.

The alignment will involve the introduction of gaps in the sequences to be aligned. in addition,
For sequences containing more or less amino acids than the protein encoded by the sequence in the sequence listing, it is understood that percent sequence identity is based on the number of identical amino acids relative to the total number of amino acids. It Thus, for example, as discussed below, sequence identity of sequences shorter than those shown in the sequence listing will be measured using the number of amino acids in the shorter sequence.

As an example, SEQ ID NOS: 1 to 51 (odd number) and SEQ ID N
OS: 53-211 was confirmed as follows. A basic Blast search was performed using the program "Blastn" and the database "nr". Blastn
Is a program of the NCBI BLAST family used to compare nucleotide query sequences against a nucleotide sequence database,
nr is all non-redundant GenBank CDS translation + PDB + Swiss
It is a nucleotide sequence database containing sProt + PIR + PRF. Two numbers, a score (bit) and an E value, will be returned after the search query is submitted. Sequences that are generally considered to be known have a score of> 100 and E <0.00
Had 1. Using the same Blast search, the nucleotide sequence encoding Exo3-118 had a score <100 or E> 0.001. Therefore, these nucleotide sequences encoding Exo3-118 were further searched using the programs "Blastn" and "dbest". The dbest database is a non-redundant Database of GenBank + EMBL +
It is a nucleotide sequence database including DDBJEST Divisions. Using these criteria, some nucleic acid sequences have scores> 100 and E <0.001; therefore, these sequences are considered novel but are also known to be suggested by the accession numbers provided. Has "some similarity" to sequences. The sequence of access numbers provided here is readily available to the technical expert.

As will be appreciated by those in the art, the nucleic acid sequences of the invention can be used to generate protein sequences. To do this, cloning of the entire gene and demonstrating its frame and amino acid sequence or assuming that the novel Exo protein has homology to some of the proteins in the database used is known. There are a variety of ways, including searching for homologies that should be compared to the sequence to give a frame. Generally, the nucleic acid sequences are input into a program that searches all three frames for homology. In a preferred embodiment this is done using the following NCBI Advanced BLAST parameters. The program is blastx or blastn. The database is nr. Input data is "Sequence in FASTA format"
The organization list is "none". The "expect" is 10.
And the filter is the default. "Description" is 500
, "Alignment" is 500, and "alignment view"
Is a pair. "Query Genetic Codes" is standard (1).
Matrix is BLOSUM62; gap exhibition cost is 11
, Per residue gap cost is 1; and the lambda ratio is .85 default. This result results in the development of the putative protein sequence. When using this program to generate preferred protein sequences, it will be appreciated that the invention provides a polypeptide encoded by each of the three frames of each nucleic acid provided herein. Therefore, when describing a protein encoded by a nucleic acid herein, the technical expert begins with the first amino acid at which the protein is encoded by the first codon of the coding region, which is not necessarily the first nucleotide in the sequence listing. Understand that there is no.

As will be appreciated by those in the art, the sequences of the present invention will include sequence errors. That is, there may be incorrect nucleotides, frameshifts, unknown nucleosides, or another type of sequence error in either sequence; however, the correct sequence falls within the homology and stringency definition here Will. In addition, as will be appreciated by those in the art, generally the first 200 bases or so of the sequence are least error-prone. In a preferred embodiment, the Exo protein is encoded by a nucleic acid that comprises the first 100 nucleotides of the sequence shown in the sequence listing and binds to an exocytosis or vesicle transport protein or fragment thereof.

The Exo proteins of the present invention may be shorter or longer than the amino acid sequences encoded by the nucleic acids displayed in the sequence listing. Thus, in one embodiment, the Exo protein can be a portion or fragment of the amino acid sequence encoded by the nucleic acid sequences provided herein. In one embodiment herein, Exo protein fragments, if they share a) at least one antigenic epitope; b) have at least the suggested sequence identity; c) and preferably exocytosis or Exo proteins are considered if they have Exo biological activity, including binding to vesicle transport proteins. In some cases where the sequence is used diagnostically, ie when determining the presence or absence of Exo protein nucleic acid, only the suggested sequence identity is required.
The nucleic acids of the invention may also be shorter or longer than the sequences in the sequence listing. Nucleic acid fragments include any portion of a nucleic acid having a previously not fully ascertained sequence provided herein; a fragment having a sequence suggested sequence identity with that portion not previously identified herein is herein The embodiment is provided.

In addition, as more fully outlined below, Exo proteins can be made that are longer than the sequences displayed in the sequence listing; eg, addition of epitopes or purified tags, addition of other fusion sequences, or additional By elucidation of coding and non-coding sequences. As described below, the green fluorescent peptide (GF
Fusions to fluorescent peptides such as P) are especially preferred.

Exo proteins also have SEQ ID NOS: 1-51, odd, and SE
Q ID NOS: 53-211, preferably those encoding Exo3-18, will be identified as being encoded by an Exo nucleic acid that hybridizes to any one of the sequences indicated. Further hybridization conditions are described below.

In a preferred embodiment, when the Exo protein is to be used for raising antibodies, the Exo protein must share at least one epitope or determinant with the full-length protein. As used herein, "epitope" or "determinant" refers to the part of a protein that produces and / or binds an antibody. Thus, in most cases, antibodies raised against the smaller Exo protein will be able to bind the full-length protein. In a preferred embodiment, the epitope is unique; that is, antibodies raised against the unique epitope show little or no cross-reactivity.
The term “antibody” refers to Fab Fab ₂ , single chain antibodies (eg Fv), chimeric antibodies, etc.
Those produced by modification of the whole antibody or using the DNA recombination technology de
antibody fragments known in the art, including those synthesized in novo.

In a preferred embodiment, antibodies to Exo are capable of reducing or eliminating the biological function of Exo, as described below. That is, Exo (or cells containing Exo) of anti-Exo antibody (polyclonal or preferably monoclonal)
Addition to will reduce or eliminate Exo activity. Generally, an activity reduction of at least 25% is preferred, fairly preferably at least about 50%, and about 95-100% reduction is particularly preferred.

The Exo antibody of the invention specifically binds to the Exo protein. In a preferred embodiment, these antibodies specifically bind the Exo protein. Here, "specifically binds" means that the antibody binds to the protein with a binding constant in the range of at least 10 ^-4 to 10 ^-6 M ^-1 , preferably in the range of 10 ^-7 to 10 ^-9 M ^-1 . Means to do. The antibody will be further described below.

In the case of nucleic acids, the overall sequence identity of nucleic acid sequences is equivalent to amino acid sequence identity but allows for degeneracy in the genetic code and codon bias of different organisms. Thus, nucleic acid sequence identity will either be lower or higher than that of protein sequences. Therefore, the sequence identity of the nucleic acid sequences compared to the nucleic acid sequences in the sequence listing is preferably 75% or more, more preferably about 80% or more, particularly preferably about 85%.
Or more, and most preferably 90% or more. In some embodiments, the sequence identity will increase from about 93% to 95% or 98%.

In a preferred embodiment, the Exo nucleic acid encodes an Exo protein.
As will be appreciated by those in the art, the degeneracy of the genetic code creates an extremely large number of nucleic acids, all of which encode the Exo proteins of the invention. Thus, upon identification of a particular amino acid sequence, one of ordinary skill in the art will be able to create any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of Exo. Let's do it.

In one embodiment, nucleic acids are identified by hybridization methods. Thus, for example, a nucleic acid that hybridizes to the nucleic acid sequences shown in the sequence listing with a high degree of stringency or its complement is the Exo gene. High stringency conditions are known in the art; eg Maniatis et al., Molecular Cloning: A Labora.
tory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biolo
gy, ed. Ausubel, et al., both of which are incorporated herein by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. For more information on nucleic acid hybridization, see Tijssen, Techniques in Bioch.
emistry and Molecular Biology Hybridization with Nucleic Acid Probes, "Ov
erview of principles of hybridization and the strategy of nucleic acid a
See ssays "(1993). In general, stringency conditions are chosen to be approximately 5-10 ° C below their thermal melting point ( _Tm ) for that particular sequence at a defined ionic strength pH.
The _Tm is in equilibrium with 50% of the probe complementary to the target hybridizing to its target sequence.
(The target sequence is present in excess at _Tm , so that 50% of the probe is occupied at equilibrium) (under defined ionic strength, pH and ionic concentration).
Stringent conditions are salt concentrations of sodium ion at pH 7.0 to 8.0 less than about 1.0, typically about 0.01 to 1.0 M sodium ion concentration (or other salts), and temperature. Is a short probe (eg 10 to 50 nucleotides)
For at least about 30 ° C and for long probes (eg, greater than 50 nucleotides) at least about 60 ° C. Stringent conditions may also be achieved with the addition of destabilizing reagents such as formamide.

In other embodiments, lower stringency conditions will be used; moderate or low stringency conditions will be used, eg as known in the art; Maniatis and A
usubel, above, and Tijssen, above.

The Exo proteins and nucleic acids of the invention are preferably recombinant. As used herein, "nucleic acid" refers to either DNA or RNA or a molecule containing both deoxy and ribonucleotides. Nucleic acid is genomic DNA, cDNA,
And oligonucleotides containing sense and antisense nucleic acids. Such nucleic acids will also include modifications in the ribose-phosphate backbone to increase the stability and half-life of such molecules in physiological environments.

Nucleic acids may include double-stranded, single-stranded, or both parts of double-stranded or single-stranded sequences. As one of ordinary skill in the art will appreciate, the single-stranded expression "Watson" also defines the sequence of the other strand "Crick"; thus, the sequences shown in the sequence listing also include the complement of that sequence. The term "recombinant nucleic acid" as used herein originally refers to i
n vitro, generally means a nucleic acid formed by the manipulation of nucleic acid with an endonuclease in a form not normally found in nature. Therefore, isolated Exo nucleic acids in a linear form or DNA molecules not normally associated with in vitro
Both expression vectors formed by ligation at o are considered recombinant for the purposes of the present invention. Once the recombinant nucleic acid is produced and reintroduced into the host cell or organism, it is non-recombinantly, ie, using the host cell's cellular machinery in vivo rather than in vitro manipulation. It will replicate; however, such a nucleic acid, once recombinantly produced and subsequently replicated non-recombinantly, is considered recombinant for the purposes of the present invention.

Similarly, a "recombinant protein" is a protein made using recombinant techniques, ie, through the expression of recombinant nucleic acids described above. Recombinant proteins are distinguished from naturally occurring proteins by at least one or more characteristics. For example, a protein will be isolated or purified from some or all of the proteins or compounds with which it is normally associated in its wild-type host, and will therefore be substantially pure. For example, an isolated protein is not associated with at least some material with which it is normally associated in its native state; preferably at least about 0.5 by weight of total protein in a given sample. %, More preferably at least approximately 5%. A substantially pure protein constitutes at least 75% by weight of the total protein, preferably at least about 80%, and particularly preferably at least about 90%. This definition is E from one organism
It also includes producing the xo protein in different organisms or host cells. on the other hand,
Through the use of inducible or highly expressed promoters, such as those that make proteins at elevated concentration levels, proteins will be made at significantly higher concentrations than normally found. On the other hand, a protein, as described below, may be in a form not normally found in nature, such as addition of epitope tags or amino acid substitutions, insertions and deletions.

Also included within the definition of Exo proteins of the invention are amino acid sequence variants. These variants fall into one or more of three types: substitutional, insertional or deletional variants. These variants are usually DN encoding the variant.
Site-directed mutagenesis of nucleotides in the DNA encoding the Exo protein using a cassette or PCR or other techniques known in the art to produce A, and then in recombinant cell culture as described above. It is created by the expression of DNA. However, the variant E having approximately 100 to 150 residues
The xo protein fragment will be generated by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by their pre-determined nature, a property that separates them from naturally occurring allelic or inter-species mutations of the Exo protein amino acid sequence. Variants typically exhibit qualitative biological activity similar to naturally occurring analogs, although variants with modified properties can also be selected, as more fully outlined below.

While the site or region for introducing an amino acid sequence mutation is predetermined, the mutation itself need not be predetermined. For example, random mutations are made at target codons or regions to optimize the performance of mutations at a given site and the Exo mutants expressed are screened for the optimal combination of desired activities. There will be. Techniques for making substitution variants at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Screening for variants is done using an Exo protein activity assay.

Amino acid substitutions are typically single residues; insertions are usually made on the order of about 1 to 20 amino acids, although fairly large ones will be tolerated. Deletions range from approximately 1 to approximately 20 residues, although in some cases they may be quite large.

Substitutions, deletions, insertions or any combination thereof can be used to arrive at the final derivative. In general, these changes are 2 to minimize alteration of the molecule.
~ 3 amino acids. However, larger changes may be tolerated under certain circumstances. When a slight modification of the characteristics of the Exo protein is desired, the following chart is generally used.

[Table 4]

[Table 5]

Substantial changes in function or immunological nature are made by selecting less conservative substitutions than those shown in Chart 1. For example, the structure of the polypeptide backbone in the altered region, eg, alpha-helix or beta-sheet structure; charge or hydrophobicity of the molecule at the target site; or substitutions that more significantly affect the bulkiness of the side chains. Will be done. Generally, the substitutions that are expected to produce the greatest change in the properties of the polypeptide are: (a) a hydrophilic residue, such as serine or threonine, is a hydrophobic residue, such as leucyl, isoleucyl,
For (or by) phenylalanyl, valyl, or alanyl
(B) cysteine or proline is substituted for (or by) any other residue; (c) a residue having a positively charged side chain, eg, lysyl,
Arginyl, or histidyl, is substituted for (or by) a negatively charged residue, eg, glutamyl, asparagyl; (d) a residue with a bulky side chain, eg phenylalanine, has no side chain. Those that are substituted for (or by) glycine.

[0260] Although the variants are also selected to modify the characteristics of the Exo protein as needed, the variants typically exhibit qualitative biological activity similar to naturally occurring analogs and similar immune responses. Will cause. On the other hand, the variant will be designed such that the biological activity of the Exo protein is modified. For example, glycosylation sites will be modified or removed. Similarly, mutations within the kinase domain and / or within the cell death domain will also be made.

Conjugate modifications of Exo polypeptides are also included within the scope of this invention. One type of conjugate modification involves reacting a target amino acid residue of an Exo polypeptide with an organic modifying reagent capable of reacting with a selected side chain or N- or C-terminal residue of the Exo polypeptide. Derivatization with a bifunctional reagent is useful, for example, for cross-linking Exo to a water-insoluble support matrix or surface for use in, for example, anti-Exo antibody purification methods or screening assays, as described in more detail below. Is. Commonly used cross-linking reagents are, for example, 1,1-bis (diazo-acetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example esters with 4-azidosalicylic acid, homobifunctional imides. Bis-N-maleimide-1,8, including esters, disuccinimidyl esters such as 3,3'-dithiobis (succinimidyl propionate)
-Difunctional maleimides such as octane and reagents such as methyl-3-[(p-azidophenyl) dithio] propioimidate.

Other modifications include deamidation of glutamine and asparagine residues to the corresponding glutamic and aspartic acids, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups in serine or threonine residues, lysine, arginine, and histidine. Methylation of side chain amino groups [TE Creighton, Proteins: Structure
and Molecular Properties, WH Freeman & Co., San Francisco, PP. 79-86 (
1983)], acetylation of N-terminal amines, and amidation of any C-terminal carboxyl groups.

Other types of conjugated modifications of Exo polypeptides included within the scope of this invention include altering the polypeptide's natural glycosylation pattern. For the purposes herein, "altering the natural glycosylation pattern" is the deletion of one or more carbohydrate moieties found in the native sequence Exo polypeptide, and / or in the native sequence Exo polypeptide. It is intended to mean the addition of one or more glycosylation sites not present in.

Addition of glycosylation sites to the Exo polypeptide will be accomplished by modification of its amino acid sequence. Modifications may be made, for example, by the addition or substitution of one or more serine or threonine residues to the native sequence Exo polypeptide (relative to the O-linkage). The Exo amino acid sequence optionally encodes an Exo polypeptide with preselected bases to generate codons that are translated into the desired amino acid, optionally through changes at the DNA level.
It will be modified by mutating NA.

Another means of increasing the number of carbohydrates on an Exo polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described, for example, in WO 87/05330, published September 11, 1987 and Aplin and
Wriston, CRC Crit. Rev. Biochem., Pp. 259-306 (1981).

Removal of carbohydrate moieties present on Exo polypeptides may be accomplished chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Techniques of chemical deglycosylation are known in the art and are described, for example, by Hakimuddin, et al., Arch. Biochem. Bioph.
ys., 259: 52 (1987) and Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides is described by Thotakura e.
can be achieved using various endo- and exo-glycosidases as described by Tal., Meth. Enzymol., 138: 350 (1987).

Another type of conjugation modification of Exo is to transfer the Exo polypeptide to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, US Pat. No. 4,640,835, 4,496,68
9,4,301,144,4,670,417,4,791,192 or 4.1
Included in the method described in No. 79,337.

The Exo polypeptides of the invention may also be modified to form chimeric molecules consisting of the Exo polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises fusing an Exo polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. Epitope tags are generally Exo
It is placed at the amino- or carboxy-terminus of the polypeptide. The presence of such epitope-tagged Exo polypeptide form can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag allows for easy purification of the Exo polypeptide by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In another embodiment, the chimeric molecule may consist of a fusion of an Exo polypeptide to an immunoglobulin or a particular region of an immunoglobulin.
For bi-binding chimeric molecules, such fusions are described in
It can be for the Fc region of a G molecule.

Various tag polypeptides and their respective antibodies are known in the art. Examples are poly-histidine (poly-his) and poly-histidine-glycine- (
Poly-his-gly) tag; influenza HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8: 2159-2165 (1988)];
c-myc tag and 8F9, 3C7, 6E10, G4, B7, and 9E
10 Antibodies to it [Evan et al., Molecular and Cellular Biology, 5: 3610-361
6 (1985)]; and herpes simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3 (6): 547-553 (1990)]. Other tag polypeptides are Flag-peptides [Hopp et al., BioTechnology, 6:12.
04-1210 (1988)]; KT3 epitope peptide [Martin et al., Scince, 255; 192-
194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem.
., 266: 15163-15166 (1991)]; and T7 gene 10 protein peptide tag [.
Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87: 6393-6397 (1990)]
including.

In certain embodiments herein, Exo proteins of the Exo family and Exo proteins from other organisms are cloned and expressed as outlined below. Therefore, probe or degenerate polymerase chain reaction (PCR) primer sequences will be used to find other related Exo proteins from humans and other organisms. As will be appreciated by those in the art, particularly useful probe and / or PCR primer sequences include the unique regions of Exo nucleic acids. As is well known in the art, preferred PCR primers are approximately 15 to approximately 35 nucleotides in length, approximately 20 to 30 nucleotides are preferred, and will contain inosine as required. The conditions for the PCR reaction are well known in the art.
Thus, as well as the sequences in the sequence listings herein, portions of these sequences are provided,
It is also understood that the unique 15 or more nucleotide portion is particularly preferred. One of ordinary skill in the art can routinely synthesize or cleave the nucleotide sequence to the desired length.

Once the Exo nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the complete Exo nucleic acid. Once isolated from its natural source, eg, contained in a plasmid or other vector or excised from it as a linear nucleic acid segment, recombinant Exo nucleic acid is used to identify or isolate other Exo nucleic acids. It can be further used as a probe. It is also a "precursor"
It can be used as a nucleic acid to make modified or mutated Exo nucleic acids and proteins. Where one or more nucleic acids overlap, one skilled in the art will delete the overlapping portion of one of the overlapping nucleic acids and combine the nucleic acids, for example by ligation, to encode, for example, a full-length or mature peptide. It is understood that such longer linear Exo nucleic acids can be formed. The same applies to the amino acid sequences of Exo polypeptides in that they can be combined to form one continuous peptide.

A variety of expression vectors are made with the nucleic acids of the invention encoding the Exo protein. The expression vector may be either a self-replicating extrachromosomal vector or a vector that integrates into the host genome. In general, these expression vectors contain transcriptional and transcriptional regulatory nucleic acid operably linked to the nucleic acid encoding the Exo protein. The term "control sequences" refers to DNA sequences necessary for the expression of operably linked coding sequences in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells have promoters, polyadenylation signals,
And it is known to use enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a DNA for a presequence or secretory leader is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that contributes to the secretion of that polypeptide; a promoter or enhancer If it affects the transcription of the sequence, it is operably linked to the coding sequence; or the ribosome binding site is operably linked to the coding sequence if it is located to promote translation. . Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader,
Meaning in close proximity and in read phase. However, enhancers do not have to be in close proximity. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to standard procedures. Transcriptional and translational control nucleic acids are generally E
It will be appropriate to the host cell used to express the xo protein; for example, transcriptional and translational control nucleic acid sequences from Bacillus are preferably used to express that Exo protein in Bacillus. Many types of suitable expression vectors, and suitable control sequences, are known in the art for various host cells.

In general, transcription and translation control sequences include, but are not limited to, promoter sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and enhancer or activator sequences. .

The promoter sequence encodes a constitutive or inducible promoter. The promoter may be either a naturally occurring promoter or a hybrid promoter. Hybrid promoters, which incorporate elements of more than one promoter, are also known in the art and are useful in the present invention.

In addition, the expression vector may consist of additional elements. For example,
The expression vector will have two replication systems and will therefore be able to be maintained in two organisms, eg mammalian or insect cells for expression and prokaryotic hosts for cloning and amplification. . Furthermore, for integrative expression vectors, the expression vector contains at least one sequence homologous to the host cell genome and preferably contains two homologous sequences flanking the expression construct.
The integrating vector will be delivered to a specific locus in the host cell by selecting the appropriate homologous sequences for inclusion in the vector. Constructs for integrating vectors are well known in the art.

In addition, in a preferred embodiment, the expression vector contains a selectable marker to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.

Preferred expression vector systems are the PCTs, both of which are incorporated herein by reference.
/ Retroviral vectors as generally described in / US97 / 01019 and PCT / US97 / 01048.

[0279] The Exo protein of the present invention is produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding the Exo protein under appropriate conditions for inducing or inducing the expression of the Exo protein. . Appropriate conditions for Exo protein expression will vary with the choice of expression vector and host cell and will be readily ascertained by one of ordinary skill in the art through routine experimentation. For example, the use of a constitutive promoter in an expression vector requires optimization of host cell growth and proliferation, while the use of an inducible promoter requires appropriate growth conditions for induction. In addition, the time of harvest is important in some embodiments. For example, the baculovirus system used for insect cell expression is a lytic virus, so harvest time selection is key to product yield.

Suitable host cells include yeast, bacteria, archaea, molds, and animal cells including insect and mammalian cells. Of particular interest is the Drosophila m
elangaster cells, Saccharomyces cerevisiae and other yeasts, Escherichia coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Red mold, BH
K, CHO, COS, and Hela cells, fibroblasts, Schwanoma cell lines,
Immortalized human myeloma and lymphoma cell lines, Jurkat cells, hepatocytes, breast cells,
They are semen, eggs, adipocytes, granule cells, adrenal chromate-staining cells, mast cells, basophils, endocrine and exocrine cells, muscle cells, eosinophils and nerve cells.

In a preferred embodiment, the Exo protein is expressed in mammalian cells. Mammalian expression systems are also known in the art and include retroviral systems.
A mammalian promoter is any D capable of binding mammalian RNA polymerase and initiating the downstream (3 ') transcription of the Exo protein coding sequence into mRNA.
NA sequence. A promoter will have a TATA box with a transcription initiation region usually located near the 5'end of the coding sequence and 25-30 base pairs upstream of the transcription initiation region. The TATA box is thought to direct RNA polymerase II to initiate RNA synthesis at the correct position. Mammalian promoters are also typically 100 to 2 upstream of the TATA box.
It will have upstream promoter elements (enhancer elements) located within 00 base pairs. Upstream promoter elements define the rate at which transcription is initiated and can act in either direction. Of particular utility as mammalian promoters are promoters from mammalian viral genes, as viral genes are often highly expressed and have a wide host range. Examples include the SV40 early promoter, mouse mammalian tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and CMV promoter.

Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are control regions located 3 ′ to the translation stop codon and thus flank the coding sequence with the promoter element. 3 of mature mRNA
The'end is formed by site-specific post-translational cleavage and polyadenylation. Examples of transcription terminators and polyadenylation signals include those derived from SV40.

Methods for introducing exogenous nucleic acids into mammalian hosts, as well as other hosts, are well known in the art and will vary with the host cell used. Examples of such techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, viral infection, liposome encapsulation of polynucleotides, and direct microinjection of DNA into the nucleus.

In a preferred embodiment, the Exo protein is expressed in bacterial systems. Bacterial expression systems are well known in the art.

[0285] A suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3 ') transcription of the Exo protein coding sequence into mRNA. Bacterial promoters will usually have a transcription initiation region located near the 5'end of their coding sequence. This transcription initiation region is typically RN
Includes A polymerase binding site and transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples of this include promoter sequences derived from sugar metabolizing enzymes such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic and hybrid promoters are also useful: for example, the tac promoter is a hybrid of trp and lac promoter sequences. In addition, bacterial promoters can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.

In addition to a functional promoter sequence, an efficient ribosome binding site is also desired. In E. coli, the ribosome binding site is the Shine-Delgarno (SD
) Referred to as a sequence and includes a start codon and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the start codon.

The expression vector will also include a signal peptide sequence that provides for secretion of the Exo protein in bacteria. As is well known in the art, the signal sequence typically encodes a signal peptide consisting of hydrophobic amino acids that directs the secretion of the protein from the cell. The protein is secreted into the growth medium (Gram-positive bacteria) or into the periplasmic space located between the inner and outer membranes of cells (Gram-negative bacteria).

The bacterial expression vector will also contain a selectable marker gene to allow for selection of transformed bacterial strains. Suitable selection genes include genes that confer bacterial resistance to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes such as those in the histidine, tryptophan and leucine biosynthetic pathways.

These components assemble into an expression vector. Expression vectors for bacteria are well known in the art, especially Bacillus subtilis, E. coli, Streptococcus.
Includes vectors for cremonis, and Streptococcus lividens.

Bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art such as calcium treatment, electroporation, and others.

In one embodiment, the Exo protein is produced in insect cells. Expression vectors for the transformation of insect cells, and especially baculovirus-based expression vectors, are well known in the art.

In a preferred embodiment, the Exo protein is produced in yeast cells. Yeast expression systems are well known in the art and include Saccharomyces cerevisiae and Candida albica.
ns and C.I. Contains expression vectors for maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillenrimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Examples of preferred promoter sequences for expression in yeast include inducible GAL1, 10
Promoter, promoter from alcohol dehydrogenase, enolase,
Glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-
3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3
-Phosphoglycerate mutase, pyruvate kinase, and acid phosphatase genes. Examples of yeast selectable markers include ADE2, HIS4, L
Examples include EU2, TRP1, and ALG7 that confers tunicamycin resistance; a neomycin phosphate transferase gene that confers G418 resistance; a CUP1 gene that enables yeast to grow in the presence of copper ions.

The Exo protein will also be a fusion protein using techniques well known in the art. Thus, for example, if the desired epitope is small, the Exo protein will be fused to a carrier protein to form an immunogen for the generation of monoclonal antibodies. Instead, the Exo protein will be a fusion protein for increased expression or for other reasons. For example, if the Exo protein is an Exo peptide, the nucleic acid encoding that peptide will be linked to other nucleic acids for expression purposes. Similarly, the Exo proteins of the present invention can be linked to protein labels such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP) and the like.

In one embodiment, the Exo nucleic acids, proteins and antibodies of the invention will be labeled. As used herein, "labeled" means having at least one element of an isotope or chemical compound attached to enable detection of the compound. Generally, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immunolabels, which may be antibodies or antigens; c) colored or fluorescent dyes. .. These labels may be inserted at any position of the compound.

In a preferred embodiment, the Exo protein is purified or isolated after expression. Exo protein depends on what other compounds are present in the sample,
It may be isolated or purified in various ways known to those skilled in the art. Examples of standard purification methods include chromatographic techniques including electrophoresis, molecular, immunological and ion exchange, hydrophobic, affinity, and reverse phase HPLC chromatography, and isoelectric chromatography. For example, Exo protein would be purified using a standard anti-Exo antibody column. Ultrafiltration and diafiltration techniques are also useful in connection with protein concentration. For general guidance on appropriate purification techniques, see Scopes, P., Protein Purification, Spri.
See nger-Verlag, NY (1982). The degree of purification required will depend on the use of the Exo protein. In some cases, purification will not be necessary.

Once expressed and, if desired, purified, Exo proteins and nucleic acids are useful in many applications.

Nucleotide sequences encoding Exo proteins (or their complements) are used in a variety of molecular biology techniques, including as probes for hybridization, in chromosome and gene mapping, and in antisense RNA and DNA. Have application. Exo protein nucleic acids will also be useful for the production of Exo proteins by the recombinant techniques described herein.

A full-length native sequence Exo protein gene or portion thereof may be isolated from the full-length Exo protein gene, or may be another gene that has the desired sequence identity to the Exo protein coding sequence (eg,
Mutations of naturally occurring Exo proteins or those encoding Exo proteins from other species) can be used as hybridization probes for cDNA libraries. Optionally, the probe length is about 2
It will be from 0 to about 50 bases. Hybridization probes may be derived from genomic sequences including the nucleotide sequences herein or the native sequence promoters, enhancer elements and introns provided herein. By way of example, the screening method may consist of isolating the coding region of the Exo protein gene using a known DNA sequence to synthesize a selected probe of about 40 bases. The hybridization probe may also be labeled with a variety of labels, including radioactive nucleotides such as ³² P or ³⁵ S or enzyme labels such as alkaline phosphatase coupled to the probe via the avidin / biotin coupling system. Good. A labeled probe having a sequence complementary to that of the Exo protein gene of the present invention is used to screen a library of human cDNA, genomic cDNA, or mRNA and the probe hybridizes to any member of such library. It can be used to measure soybean.

Probes may also be used in PCR technology to generate sequence pools for confirmation of closely related Exo protein coding sequences.

The nucleotide sequence encoding the Exo protein can also be used to construct hybridization probes for mapping of the gene encoding the Exo protein and genetic analysis of individuals with a genetic disorder. The nucleotide sequences provided herein have been mapped to chromosomes or specific regions of chromosomes using known techniques, such as in situ hybridization, linkage analysis for known chromosomal markers, and library hybridization screening. Good.

Nucleic acids encoding the Exo proteins or modified forms thereof can also be used to generate transgenic or "knockout" animals, which, conversely, are useful in the development and screening of therapeutically valuable reagents. A transgenic animal (eg, mouse or rat) is an animal that has cells that contain the transgene into which the transgene has been introduced prior to birth in the animal or its ancestors, eg, at the fetal stage. The transgene is the DNA that is integrated into the genome of the cells from which the transgenic animal is bred. In one embodiment, the cDNA encoding the Exo protein can be used to clone genomic DNA encoding the Exo protein according to established techniques, and the genomic sequence used to Transgenic animals can be generated that contain cells that express the DNA. Methods for generating transgenic animals, particularly animals such as mice and rats, have become convenient in the art and are described, for example, in US Pat. Nos. 4,736,866 and 4,870,009. Typically, particular cells will be targeted for Exo protein transfer gene insertion by tissue-specific enhancers. E introduced into the germ line of the animal in the fetal state
Transgenic animals containing a copy of the transgene encoding the xo protein can be used to investigate the effect of increased expression of the desired nucleic acid. Such animals are
For example, it can be used as a laboratory animal for reagents believed to confer protection from the pathological conditions associated with its overexpression. In accordance with this aspect of the invention, the reduced incidence of that pathological condition compared to untreated animals in which the animal is treated with the reagent and carries the transplant gene is a potential therapeutic intervention for that pathological condition. Would suggest.

Alternatively, using a non-human homologue of the Exo protein, a cognate set between the endogenous gene encoding the Exo protein and the modified genomic gene encoding the Exo protein introduced into animal fetal cells. As a result of the alteration, "knockout" animals can be constructed that have a missing or modified gene encoding the Exo protein. For example, a cDNA encoding an Exo protein can be used to clone genomic DNA encoding an Exo protein according to established techniques. A portion of the genomic DNA encoding the Exo protein is
It can be deleted or replaced with another gene, such as the gene encoding a selectable marker that can be used for monitor integration. Typically, the unmodified flanking DNA (
Several kilobases (both at the 3'and 5'ends) are included in the vector [eg,
For a description of cognate recombinant vectors, see Thomas and Capecchi, Cell, 51: 503.
(1987)]. Introducing the vector into fetal stem cells (for example, by electroporation)
) And select cells in which the introduced DNA has cognately recombined with the endogenous DNA [see, eg, Li et al., Cell, 69: 915 (1992)]. The selected cells are then injected into the blastula of an animal (eg, mouse or rat) to form an assembled chimera [eg, Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, EJ Robertson, ed. (IRL, Oxford, 1987), pp. 113-152].
The chimeric fetus is implanted into a suitable pseudopregnant female foster animal and the fetus is delivered, resulting in the production of a "knockout" animal. Progeny that harbor the cognately recombined DNA in their germ cells are identified by standard techniques, and all cells of the animal are bred to carry the cognately recombined DNA. Can be used for Knockout animals can be characterized, for example, for their ability to defend against certain pathological conditions and for the development of pathological conditions due to their lack of Exo protein polypeptides.
It is understood that cell-based knockout or knockin systems can also be made and used in accordance with the present disclosure.

It is understood that the model described here can vary. For example, a "knock-in" model can be created, or the model can be cell-based rather than animal model.

Nucleic acids encoding Exo polypeptides, antagonists or agonists may also be used in gene therapy. In gene therapy applications, the gene may have a therapeutically effective gene product in vivo, for example to replace a defective gene.
Introduced into cells to achieve synthesis. "Gene therapy" means convenient gene therapy and therapeutically effective DNA or RN in which the sustained effect is achieved by simple treatment.
It includes two of gene therapy drug administration, including temporary or repeated administration of A. Antisense RNAs and DNAs can be used as therapeutic agents to block the expression of genes in vivo. Despite its low intracellular concentration caused by short antisense oligonucleotides by suppressing their uptake by the cell membrane,
It has already been shown that it can be imported into cells that act as inhibitors. [Zam
ecnik et al., Proc. Natl. Acad. Sci. USA 83, 4143-4146 (1986)]. The oligonucleotide may be modified to enhance its uptake, for example by replacing its negatively charged phosphodiester group with an uncharged group.

There are a variety of techniques available for introducing nucleic acids into living cells. The technique involves the transfer of nucleic acids to cultured cells in vitro or to cells of the intended host in vitro.
It depends on whether or not it is transplanted in vivo. Suitable techniques for in vitro implantation of nucleic acids into mammalian cells include liposomes, electroporation, microinjection, cell fusion,
Including DEAE-dextran, calcium phosphate precipitation method and the like. Currently preferred in
In vivo gene transfer technology is based on viral (typically retroviral) vectors and viral mantle liposome-mediated transfection [Dzau et al., Trends i.
Biotechnology 11, 205-210 (1993)]. In some circumstances it may be desirable to provide a source of nucleic acid with reagents that target the target cell, such as cell surface membrane proteins or antibodies specific to the target cell, receptor ligands on the target cell, and the like. When using liposomes, proteins that bind to cell surface membrane proteins involved in endocytosis are targeted and / or for example, proteins that internalize into capsid proteins or fragments thereof directed to specific cell lineages, circuits. It may also be used to promote the uptake of proteins that target intracellular localization and increase intracellular half-life, such as antibodies to. Techniques for receptor-mediated endocytosis are described, for example, in Wu et al., J. Biol. Chem. 26.
2, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87,
3410-3414 (1990). For a review of gene regulation and gene therapy protocols, see Anderson et al., Science 256, 808-813 (1992).

In a preferred embodiment, cells containing Exo proteins, nucleic acids, modified proteins and native or modified Exo proteins are used in screening assays. Identification of this key exocytosis protein allows the design of an agent screening assay for compounds that modulate Exo activity.

A screen may be designed to first find candidate agents that bind to the Exo protein and then be used in an assay to assess the ability of these agents to modulate the Exo activity of the candidate agent. Thus, as the skilled artisan will appreciate, there are a variety of different assays that can be performed; binding assays and activity assays.

Therefore, in a preferred embodiment, the method comprises a combination of Exo protein and a candidate bioactive agent and measuring the binding of the candidate agent to the Exo protein. Rodents (mouse, rat, hamster, guinea pig, etc.), livestock (
In a preferred embodiment human Exo protein is used, although other mammalian proteins may also be used, including bovine, ovine, porcine, equine, etc.) and primates. These latter embodiments are preferred in the development of animal models of human disease. As outlined herein, in some embodiments, deletion E as outlined above is used.
Variant or inducible Exo proteins may be used, including xo proteins.

The term “candidate bioactive agent” or “exogenous compound”, as used herein, refers directly or directly to the biological activity of Exo, eg, proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides and the like. Refers to any molecule capable of being indirectly modified. Generally, multiple assay mixtures are run in equilibrium with different concentrations of agent to give different responses to different concentrations. Typically, one of these concentrations serves as a negative control, ie at zero concentration or below the level of detection.

Candidate agents typically include organic molecules, preferably small organic molecules having a molecular weight of 100 or more and about 2,500 or less daltons, but encompass many classes of compounds. Candidate agents consist of functional groups necessary for structural interaction with proteins, especially hydrogen bonding, and are typically at least amine, carbonyl, hydroxyl or carboxyl groups, preferably at least two chemical groups. It has a functional group. Candidate agents are often substituted with one or more of the above functional groups,
It consists of cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic ring structures. Candidate agents are also found in biomolecules including peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic and natural compounds. For example, numerous means are available for random and controlled synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. On the other hand, natural product libraries in the form of bacterial, fungal, plant and animal extracts are available or readily produced. In addition, natural and synthetically produced libraries and compounds are convenient chemical, physical,
And easily modified through biochemical means. Known pharmacological agents will be subjected to controlled or random chemical modifications such as acylation, alkylation, esterification, amidation to produce structural analogs.

In a preferred embodiment, the candidate bioactive agents are proteins. here"
"Protein" means at least two covalently joined amino acids, which includes proteins, polypeptides, oligopeptides, and peptides. Proteins are derived from naturally occurring amino acids and peptide bonds, or from synthetic peptidomimetic structures. Thus, “amino acid”, or “peptide residue”, as used herein, refers to both naturally occurring and synthetic amino acids, eg, homophenylalanine, citrulline and norleucine are the objects of the invention. For the purpose of being considered an amino acid, "amino acid" also comprises imino acid residues such as proline and hydroxyproline, the side chains of which may be in either the (R) or (S) conformation. In a preferred embodiment, the amino acid is (S) or L-
Conformation. If non-natural side chains are used, non-amino acid substituents may be used, for example, to prevent or delay degradation in vivo.

In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cell extracts containing proteins or random or controlled digests of proteinaceous cell extracts may be used. In this way, a library of prokaryotic and eukaryotic proteins can be transformed into Ex.
will be created for screening for o. Particularly preferred in this embodiment are bacterial, fungal, viral and mammalian proteins, the latter being preferred and human proteins being especially preferred.

In a preferred embodiment, the candidate bioactive agent is a peptide consisting of about 5 to about 30 amino acids, preferably about 5 to about 20 amino acids, and about 7 to about 15 amino acids.
Amino acids are particularly preferred. Peptides may be digests of naturally occurring proteins as outlined above, random peptides or "biased" random peptides. "Randomized" or grammatical synonyms herein means that each nucleic acid and peptide is essentially composed of random nucleotides and amino acids, respectively. In general, these random peptides (or nucleic acids, discussed below) are chemically synthesized, so they will insert any nucleotide or amino acid at any position. The synthetic steps are designed to generate randomized proteins or nucleic acids, permitting the formation of all or most possible combinations over their sequence length, thus forming a library of randomized candidate bioactive proteinaceous agents.

In one embodiment, the library is well randomized and has no sequence preference or consistency at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are kept constant or a limited number of possibilities are selected. For example, in a preferred embodiment, the nucleotide or amino acid residues are random within a defined class, such as hydrophobic amino acids, hydrophilic amino acids, sterically biased (either small or large) residues. Cysteine for cross-linking, SH-3
Proline for domains, serine for phosphorylation, threonine, tyrosine,
Alternatively, it is directed to production such as histidine or purine.

In a preferred embodiment, the candidate bioactive agent is a nucleic acid. "Nucleic acid" or "oligonucleotide" or grammatical synonyms herein means at least two nucleotides joined together covalently. Nucleic acids of the invention will generally include phosphodiester bonds, but as outlined below, in some cases nucleic acid analogs with alternating backbones are included, consisting of, for example, phosphoramide ( Beaucage et al., Tetrahedron 49 (10): 1925 (1993) and references therein; Letsinger et al., J Org. Chem. 35: 3800 (1970); Sprinzi et al.,
Eur. J. Biochem. 81: 579 (1977); Letsinger et al., Nucl. Acids Res. 14: 3.
487 (1986); Sawai et al., Chem. Lett., 805 (1984), Letsinger et al., J.
Am. Chem. Soc. 110: 4470 (1988); and Pauwels et al., Chemica Scripts 26: 1.
419 (1986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19: 1437.
(1991); and US Pat. No. 5,644,048), phosphorodithioate (Briu et.
al., J. Am. Chem. Soc. 111: 2321 (1989)), O-methyl phosphoramidite bond (Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford
(See University Press), and peptide nucleic acid backbone and binding (Egh
olm, J. Am. Chem. Soc. 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl.
.31: 1008 (1992); Nielsen, Nature, 365: 566 (1993); Carlsson et al., Natur
e 380: 207 (1996), all of which are incorporated herein by reference). Examples of other similar nucleic acids include positive backbone (Denpcy et al., Proc. Natl.
Acad. Sci. USA 92: 6097 (1995); non-ionic backbone (US Pat. No. 5,38.
6,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30
: 423 (1991); Letsinger et al., J. Am. Chem. Soc. 110: 4470 (1988); Letsin
ger et al., Nucleoside & Nucleotide 13: 1597 (1994); Chapters 2 and 3, AS
C Symposium Series 580, "Carbohydrate Modifications in Antisense Researc
h ", Ed. YS Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Med
icinal Chem. Lett. 4: 395 (1994); Jeffs et al., J. Biomolecular NMR 34:17.
(1994); Tetrahedron Lett. 37: 743 (1996)) and US Pat. No. 5,235,033.
5,034,506 and Chapters 6 and 7, ASC Symposium Series 580, "
Carbohydrate Modifications in Antisense Research ", Ed. YS Sanghui and
Non-ribose backbones, including those described in P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also within the definition of nucleic acids (J
See enkins et al., Chem. Soc. Rev. (1995) pp169-176). Some nucleic acid analogs are described in Rawls C & E News June 2, 1997 Page 35. All of these references are incorporated herein by reference. These modifications of the ribose phosphate backbone may be made to facilitate the addition of additional sites such as labels or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, a mixture of different nucleic acid analogs and a mixture of naturally occurring nucleic acids and analogs may be made. The nucleic acid may be single stranded or double stranded, as specified, or may include portions of both double stranded or single stranded sequence. Nucleic acid is DN
A, both genomic and cDNA, RNA or hybrid where the nucleic acid is any combination of deoxyribo and ribonucleotides, as well as uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, It includes any combination of bases including isoguanine and the like.

As described above generally for proteins, the nucleic acid candidate bioactive agent may be a naturally occurring nucleic acid, a random nucleic acid, or a “biased” random nucleic acid. For example, a prokaryotic or eukaryotic genomic digest can be used as outlined above for proteins.

In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.

The assay provided uses the Exo protein as defined herein. In one embodiment, a portion of the Exo protein is used. In a preferred embodiment, Ex
A portion having o activity is used. Exo activity is described further below and further below as GS27, rab7, rab9, snap-23, rab3a,
rab11, rab3d, rab5, alpha-snap, unc18-1,
Includes binding activity to vamp3 or Exo protein modulators.
In addition, the assays described herein may use isolated Exo protein or cells that make up Exo protein.

In general, in a preferred embodiment of the methods herein, the Exo protein or candidate agent is non-diffusible on an insoluble support with a separate sample receiving area (eg, microtiter plate, array, etc.). Bind to. The insoluble support may be made up of any constituent to which each component binds, is easily separated from the soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and may be of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. They are typically glass, plastic (eg polystyrene), polysaccharides, nylon or nitrocellulose, Teflon®, etc. Microtiter plates and arrays are particularly convenient because the majority of assays can be performed simultaneously with small amounts of reagents and samples. In some cases, magnetic beads and the like may also be mentioned.
The particular manner in which the components are combined is not particularly critical, so long as it is compatible with the reagents and the overall method of the invention, retains the activity of the components, and is non-diffusible. Preferred methods of attachment include the use of antibodies (which do not sterically block either the ligand binding site or the activation sequence when the protein binds to the support), “sticky” or ionic supports. Direct binding, chemical cross-linking, synthesis of proteins or agents on the surface and the like. In some embodiments, G
S27 can be used. In another embodiment, rab7, rab3a, rab3d
, Snap 23, rab9, rab5, alpha snap, rab11,
The use of unc18-1 or vamp3 may be mentioned. After binding the protein or agent, excess unbound material is removed by washing. The sample receiving area may then be blocked by incubation with bovine serum albumin (BSA), casein or other innocuous protein or other component. Screening assays that do not use a solid support are also included in this invention.

In a preferred embodiment, Exo protein is bound to a support and candidate bioactive agents are added to the assay. Alternatively, the candidate agent is bound to the support and the Exo protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified by screening chemical libraries, peptide analogs, and the like. Of particular interest are screening assays for agents that are of low toxicity to human cells. A wide variety of assays can be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, protein binding immunoassays, functional assays (phosphorylation assays, etc.). Etc.

The determination of binding of a candidate bioactive agent to the Exo protein may be done in a number of ways. In a preferred embodiment, the candidate bioactive agent is labeled and binding is measured directly. For example, to do this, all or part of the Exo protein is attached to a solid support, a labeled candidate agent (eg, a fluorescent label) is added, excess reagent is washed away, and the label is solid. It may be measured whether it is present on the carrier. Various blocking and washing steps may be utilized as are known in the art.

By “labeled” herein is meant that the compound is labeled either directly or indirectly with a label, such as radioisotopes, fluorescent substances,
It provides a detectable signal for enzymes, antibodies, particles such as magnetic particles, chemiluminescent substances, specific binding molecules and the like. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and anti-digoxin. For specific binding members, the complementary member will typically be labeled with a molecule that provides for detection, according to known procedures, as outlined above. This label may give a detectable signal, either directly or indirectly.

In some embodiments, only one component is labeled. For example, the protein (or proteinaceous candidate agent) may be labeled at the tyrosine position using ¹²⁵ I or with a fluorophore. Alternatively, one or more components may be labeled with separate labels; for example, ¹²⁵ I is used for proteins and fluorophores are used for candidate agents.

In a preferred embodiment, binding of candidate bioactive agents is measured by use of competitive binding assays. In this aspect, a competitor is a binding moiety known to bind a target molecule (eg, Exo), such as an antibody, peptide, binding partner, ligand, etc. In a preferred embodiment, the competitor is GS27, rab
7, rab9, snap-23, rab3a, rab11, rab3d, rab
5, alpha-snap, unc18-1 or vamp3. Under certain circumstances, there may be competitive binding, such as between the candidate agent and the binding component, but the binding component replaces the candidate agent. Using this assay, Exo protein and GS27, rab7, rab9, snap-23, rab3a, ra
Candidate agents that interfere with the binding between bl1, rab3d, rab5, alpha-snap, unc18-1 or vamp3 can be measured.

In one embodiment, candidate bioactive agents are labeled. Either the candidate bioactive agent or the competitor, or both, is first added to the protein for a time sufficient to allow binding, if any. Incubations may be performed at any temperature that promotes optimal activity, typically between 4 and 40 ° C. The incubation period is chosen for optimal activity, but may also be optimized to facilitate rapid, high throughput screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is typically removed or washed away. The second component is then added and the presence or absence of the labeled component is followed to indicate binding.

In a preferred embodiment, the competitor is added first, followed by the candidate bioactive agent. Competitor displacement is an indication that a candidate bioactive agent has bound to the Exo protein and is therefore capable of binding and potentially modulating the activity of the Exo protein. In this aspect, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of the label in the wash solution indicates displacement of the agent. Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive agent is added first, followed by incubation and washing, followed by the competitor. The absence of binding by the competitor would indicate that the bioactive agent binds Exo protein with a higher affinity. Thus, if the candidate bioactive agent is labeled, the presence of the label on the support, along with the lack of competitor binding, would indicate that the candidate agent is capable of binding the Exo protein.

In a preferred embodiment, the method comprises a differential screen to identify active agents capable of modulating the activity of Exo protein. In this embodiment, these methods consist of binding Exo protein and competitor in a first sample. The second sample consists of the candidate bioactive agent, Exo protein and competitor. Competitor binding was measured for both samples, and changes or differences in binding between the two samples indicate the presence of agents capable of binding the Exo protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample compared to the first sample, the agent is capable of binding the Exo protein.

Alternatively, a preferred embodiment utilizes a differential screen to identify drug candidates that bind to the native Exo protein but not the modified Exo protein. The structure of the Exo protein will be modeled and used in rational drug design to synthesize agents that interact with that site. Drug candidates that act on Exo bioactivity are also identified by screening drugs for their ability to either enhance or decrease the activity of the protein.

Positive and negative controls may be used in the assay. Preferably, all control and test samples are run at least 3 times to obtain statistically meaningful results. Incubation of all samples is for a time sufficient for the agent to bind to the protein. After incubation, all samples are washed to remove non-specifically bound material and bound, generally labeled agent is measured. For example, if a radiolabel is used, the sample is counted in a scintillation counter to determine the amount of bound compound.

A variety of other reagents may be included in the screening assay. They include reagents such as salts, neutral proteins, eg albumin, detergents, etc., which are used to promote protein-protein binding and / or reduce non-selective or background interactions. Will be done. Alternatively, reagents that otherwise improve the efficiency of the assay may be used, such as protease inhibitors, nuclease inhibitors, antibacterial agents and the like. The mixture of components may be added in any order that provides for the requisite binding.

The components provided herein for the assays provided herein may also be combined to form a kit. The kit can be based on the use of proteins and / or nucleic acids encoding Exo proteins. Assays based on the use of nucleic acids are described further below.

Screening for agents that modulate the activity of Exo may also be done. In a preferred embodiment, the method of screening for bioactive agents capable of modulating the activity of Exo comprises adding a candidate bioactive agent to a sample of Exo and altering the biochemical activity of Exo, as described above. Measuring.
"Modulation of Exo activity" includes increased activity, decreased activity, or changes in the type or kind of activity that is present. Therefore, in this embodiment, the candidate agent must bind to Exo (although this may not be necessary) as well as alter its biological or biochemical properties, as defined herein. This method generally involves in vitro screening methods and cell in vitro response to changes in the presence, distribution, activity or amount of Exo, as outlined above.
Includes both vo screening.

Therefore, in this embodiment, the method comprises binding a sample of Exo with a candidate bioagent and assessing its effect on exocytosis. As used herein, "Exo activity" or grammatical equivalents refers to the biological activity of Exo and includes, but is not limited to, the ability to affect exocytosis, secretion and / or vesicle transport. Exocytosis, secretion and /
Alternatively included in the vesicle transport activity are exocytosis, secretion and / or
Or the regulation or involvement of steps therein such as docking, fusion and targeting activity of proteins involved in the overall pathway of vesicle trafficking. In one embodiment, vesicular refers to any vesicle, including synaptic or secretory granules and vesicles, which are involved in exocytosis, endocytosis or trans-Golgi networks. In one embodiment, ex
o activity includes GTPase activity or regulation thereof. Here, one exo activity is GS27, lab7, lab9, snap-23, lab3a, lab11, r.
ab3d, rab5, alpha-snap, unc18-1 and vamp3.
Binding to at least one protein selected from the group consisting of: Other exo activities are GS27, lab7, lab9, snap-23, lab3a,
It includes the activities of rab11, rab3d, rab5, alpha-snap, unc18-1 and vamp3 and their regulation.

In one preferred embodiment, the activity of Exo protein is increased; in another preferred embodiment, the activity of Exo protein is decreased. Thus, bioactive agents that are antagonists will be preferred in some embodiments, and bioactive agents that will be agonists will be preferred in other embodiments.

In a preferred embodiment, the invention provides a method of screening for bioactive agents capable of modulating the activity of Exo protein. This method
As defined above, consisting of adding a candidate bioactive agent to cells consisting of Exo protein. Preferred cell types include almost any cell. This cell contains a recombinant nucleic acid encoding an Exo protein. In a preferred embodiment, the screening method tests a library of candidate agents on a plurality of cells, as described above.

In certain embodiments, the assay comprises exposing cells to an exocytosis agent, which will induce exocytosis in control cells, ie cells of the same type, but encodes Exo. Does not contain exogenous nucleic acid. Suitable exocytosis agents are known in the art, and it seems ionomycin and Ca ⁺⁺ ionophore A23187 without limitation including Ca ⁺⁺ but not limited to. Alternatively, cells may be exposed to conditions that normally result in exocytosis, and changes in normal exocytosis progression are measured. Instead, Exo nucleic acid is normally introduced into cells under exocytosis and thus the change (eg, exocytosis) is measured. Optionally, the bacterium does not normally undergo exocytosis, and the introduction of the candidate agent causes exocytosis.

In this way, candidate agent effects on exocytosis are then evaluated.

Detection of exocytosis may be performed as would be appreciated by one of skill in the art. In one embodiment, an exocytosis indicator is used. Suitable exocytosis labels include, but are not limited to, annexins. Thus, these agents are used as affinity ligands, attached to solid supports such as beads, surfaces, etc., and used to elicit cells undergoing exocytosis. Similarly, these agents can be linked to a fluorescent dye such as PerCP and then used as the basis for fluorescence-activated cell sorting (FACS) separations. In addition, using FACS or other optical methods,
Exocytosis activity and its regulation can be detected based on light absorption, dye uptake and release, granule enzyme activity and quantification of granule-specific proteins.

In this way, bioactive substances are identified. Exo is a pharmacologically active compound
It can enhance or interfere with protein activity. The desired pharmacologically active compound may be administered to the host in a physiologically acceptable carrier, as described above. The agent may be administered in various ways orally, parenterally, eg subcutaneously, intraperitoneally, intravascularly, etc. The compound may be formulated in a variety of ways depending on the mode of administration. The concentration of therapeutically active compound may vary from about 0.1-100% by weight.

The pharmaceutical composition can be formulated in various dosage forms such as granules, tablets, pills, suppositories, capsules, suspensions, ointments, lotions and the like. Compositions containing therapeutically active compounds can be prepared with pharmaceutical grade organic or inorganic carriers and / or excipients suitable for oral and topical use. Excipients known in the art include aqueous media, vegetable and animal oils and fats. Stabilizer,
Wetting and emulsifying agents, salts that alter the osmotic pressure, buffers to maintain proper pH values and skin permeation enhancers can be used as adjuvants.

Without being bound by theory, Exo is a protein important for exocytosis. Thus, diseases based on mutant or mutant Exo genes will be determined. In one embodiment, the invention provides a method of identifying a cell containing a mutant Exo gene, which comprises determining all or part of the sequence of at least one endogenous Exo gene in the cell. This may be done using any number of sequencing techniques, as will be appreciated by those in the art. In a preferred embodiment, the invention comprises at least one Ex of an individual.
There is provided a method of identifying an Exo genotype in an individual comprising determining all or part of the sequence of the o gene. This is typically done on at least one tissue of an individual, and may include the evaluation of multiple tissues or different samples of the same tissue. The method involves comparing the sequenced Exo gene with a known Exo gene, the wild type gene.

The sequence of all or part of the Exo gene is then compared to the sequence of the known Exo gene to determine what differences exist. This can be done with any number of known sequence identity programs such as Bestfit. In a preferred embodiment, the presence of sequence differences between a patient's Exo gene and a known Exo gene is indicative of a propensity for a condition or condition, as outlined herein.

The present findings regarding the role of Exo in exocytosis thus provide a method of inducing or preventing exocytosis in cells. In a preferred embodiment, the Exo protein, and in particular the Exo fragment, is used for the study or treatment of conditions mediated by exocytosis, ie for diagnosing, treating or preventing diseases mediated by exocytosis. It is useful. Thus,
"Diseases mediated by exocytosis" or "conditions" include conditions involving both insufficient or excessive exocytosis, transport and / or secretion via the secretory pathway, including asthma, allergies and Chediak-Higa.
Includes the release of inflammatory mediators from mast cells, including shi syndrome. In addition, control of neurotransmitter release may be used to treat Alzheimer's disease, Parkinson's and Huntington's pathology and some schizophrenia, so that in some cases they are mediated by exocytosis. It can be included in the disease. In other cases, fertilization and feeding disorders may be included as medical conditions treatable with the components provided and / or identified herein. In addition to this, certain diabetes, digestive and wound healing diseases can be diseases mediated by exocytosis.

[0346] Thus, in one embodiment, a method of modulating exocytosis in a cell or organism is provided. In one embodiment, the method comprises administering to the cell an anti-Exo antibody that reduces or eliminates the biological activity of the endogenous Exo protein. Instead, this method consists of administering a recombinant nucleic acid encoding an Exo protein into a cell or organism. This may be accomplished in any numbered fashion, as will be appreciated by those skilled in the art. In a preferred embodiment, the activity of Exo is increased by increasing the amount of Exo in the cell, for example using known gene therapy techniques, eg endogenous Exo.
Is overexpressed or by administering the gene encoding Exo. In a preferred embodiment, the gene therapy technique is an exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT / US93 / 03868, which is incorporated herein by reference in its entirety. Including the inclusion of

In one embodiment, this invention provides a method of diagnosing an exocytosis-related condition in an individual. This method consists of measuring the activity of Exo in tissues from an individual or patient, which involves measuring the amount of specific activity of Exo. This activity is compared to the activity of Exo from either unaffected second individuals or unaffected tissues from the first individual. When these activities differ, the first individual may be at risk for a disease mediated by exocytosis.

The proteins and nucleic acids provided herein can also be used for screening purposes, where protein-protein interactions of Exo proteins can be confirmed. Genetic systems have been described to detect protein-protein interactions. The first work was done in the yeast system, the "yeast two-hybrid" system. The basic system requires protein-protein interactions to initiate transcription of reporter genes. The next work was done on mammalian cells. Fields et al., Nature 340: 245 (1989); Vasavada et al., PNAS US
A 88: 10686 (1991); Fearon et al., PNAS USA 89: 7958 (1992); Dang et al.
, Mol. Cell. Biol. 11: 954 (1991); Chien et al., PNAS USA 88: 9578 (1991).
); And US Pat. No. 5,283,173, 5,667,973, 5,468,614.
See 5,525,490, and 5,637,463. A preferred system is described in Serial No. 09 / 050,863, filed Mar. 30, 1998, entitled "Mammalian Protein Interaction Cloning System". A particularly useful shuttle vector for use in conjunction with these systems is S
serial No. 09 / 133,944, filed Aug. 14, 1998, entitled "Shuttle Vector".

Generally, two nucleic acids are transformed in cells, one of which is GS27.
, Lab7, lab9, snap-23, lab3a, lab11, lab3d
, Rab5, alpha-snap, unc18-1, vamp3 or a part thereof is a "bait", and the other one encodes a test candidate. An indicator such as a fluorescent protein will be expressed only when the two expression products bind to each other. Regarding the expression of the indicator, the test candidates are GS27, lab7, lab9, snap-23, lab3a, r.
It has been shown to bind to abl1, rab3d, rab5, alpha-snap, unc18-1 or vamp3 and is identified as an Exo protein. The same system and the identified Exo protein can be used to perform the reverse. That is, the Exo proteins provided herein can be used to identify new decoys or agents that interact with the Exo proteins. In addition to this, a two-hybrid system can be used, where test candidates are
In addition to decoys and Exo proteins encoding nucleic acids, GS27, r
ab7, lab9, snap-23, lab3a, lab11, lab3d, r
ab5, alpha-snap, unc18-1 or vamp3 and Ex
o Agents that interfere with decoys such as proteins are measured.

In one embodiment, mammalian two-hybrid systems are preferred. Mammalian systems provide post-translational modifications of proteins that will contribute significantly to their ability to interact. In addition, the mammalian two-hybrid system can be used with a wide variety of mammalian cell types to mimic specific protein regulation, induction, processing, etc. within a particular cell type. For example, proteins involved in pathologies such as those described above could have been tested in cells of related diseases. Similarly, for testing random proteins, assaying them under relevant cellular conditions will give the highest positive results. In addition, mammalian cells are tested under a variety of experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokines, cellular and chemical stimuli, etc. However, these will contribute to conditions where they can lead to protein-protein interactions, especially those involved in exocytosis, secretory pathways and / or vesicle trafficking.

Assays for expression and Exo activity in various cell types have been described above. Assays for activities that affect exocytosis, secretory pathways and / or vesicle trafficking were performed to determine GS27, rab7, rab9, snap-2.
3, lab3a, lab11, lab3d, lab5, alpha-snap,
Confirming the activity of the Exo protein identified by sequence identity / similarity or binding to unc18-1 or vamp3, and a lead identified further as a modulator of exocytosis, secretion and / or vesicle trafficking The activity of the compound can be confirmed.

Assays involving binding, such as the two-hybrid system, may consider non-specific binding proteins (NBS).

In one embodiment, the Exo proteins of the invention can be used to raise polyclonal and monoclonal antibodies against the Exo proteins, which are useful as described herein. Similarly, the Exo protein can be linked to an affinity chromatography column using standard techniques. The Exo antibody can then be purified using these columns. In a preferred embodiment, it is raised to an epitope unique to the Exo protein; that is, the antibody exhibits little or no cross-reactivity to other proteins. These antibodies are used in many applications. For example, the Exo antibody may be linked to a standard affinity chromatography column and used to purify the Exo protein as described further below. Antibodies also bind specifically to the Exo protein and may therefore be used as blocking polypeptides, as outlined above.

The anti-Exo protein antibody may consist of a polyclonal antibody. Methods of making polyclonal antibodies are known to those of skill in the art. Polyclonal antibodies can be raised in mammals, for example, by one or more injections of an immunizing agent and, optionally, an adjuvant. Typically, the immunizing agent and / or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may contain the Exo protein polypeptide or a fusion protein thereof. It would be useful to conjugate the immunizing agent to a protein known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin and soybean trypsin inhibitor. Examples of adjuvants that may be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid-synthetic trehalose dicorynomycolate). Immunization protocols can be selected by those of skill in the art without undue experimentation.

The anti-Exo protein antibody may alternatively be a monoclonal antibody. Monoclonal antibodies can be made by the hybridoma method as described by Kohler and Milstein, Nature, 256: 495 (1975). In the hybridoma method, the ability of a mouse, hamster, or other suitable host animal to be immunized typically with an immunizing agent to produce or produce an antibody that will specifically bind to the immunizing agent. There are provoked lymphocytes. Alternatively, lymphocytes may be immunized in vitro.

The immunizing agent will typically contain an Exo protein polypeptide or a fusion protein thereof. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph nodes are used if non-human mammalian sources are desired. Or either. The lymphocytes are then fused with an immortalizing cell line using a suitable fusing agent such as polyethylene glycol to form hybridoma cells [Goding, Monoclonal An
tibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
Immortalized cell lines are usually transformed mammalian cells, especially myeloma of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells may be cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cell lacks the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT),
The medium for hybridomas contains hypoxanthine, aminopterin and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. . More preferred immortalized cell lines are mouse myeloma cell lines, which are described, for example, in the Salk Institute Cell Distribution Center, San Diego, Californ.
Obtained from ia and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines have also been described for human monoclonal antibody production [Kozbor, J. Immunol., 133: 3001 (198
4); Brodeur et al., Monoclonal Antibody Production Techniques and Applic
ations, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the Exo protein. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is immunoprecipitation or radioimmunoassay (RIA) or eraser (EL).
It is measured by an in vitro binding assay such as IZA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody is
For example, it can be measured by the Scatchard analysis of Munson and Polland, Anal. Biochem., 107: 220 (1980).

Once the desired hybridoma cells have been identified, clones are subcloned by limiting dilution procedures and expanded by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle Medium and RPM.
I-1640 medium is mentioned. Alternatively, hybridoma cells may be grown in vivo as ascites in mammals.

The monoclonal antibody secreted from the subclone can be prepared from a medium or ascites fluid by a conventional immunoglobulin purification operation method such as protein α-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. Can be isolated or purified by.

Monoclonal antibodies can also be produced by recombinant DNA methods as described in US Pat. No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily isolated and sequenced in a conventional manner (eg, using an oligonucleotide probe capable of specifically binding to the genes encoding the light chain and heavy chain of mouse antibody). You can decide. The hybridoma cells of the invention serve as a preferred source of such DNA. After isolation, the DNA is placed in an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins in situ, Monoclonal antibodies are synthesized in transgenic host cells. The DNA may also replace, for example, the sequences encoding the constant domains of human heavy and light chains with homologous mouse sequences (US Pat. No. 4,816,816).
, 567; Morrison et al., Supra), or may be modified by covalently linking all or part of the sequence encoding the non-immunoglobulin polypeptide to the sequence encoding the immunoglobulin. Such a non-immunoglobulin polypeptide may replace the constant domain of the antibody of the present invention, or may replace the variable domain of the antigen binding site of the antibody of the present invention to produce a chimeric divalent antibody.

The antibody may be a monovalent antibody. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chains and modified heavy chains.
The heavy chain may be truncated generally at any site in the Fc region to prevent heavy chain crosslinking. Alternatively, related cysteine residues may be replaced or deleted with other amino acid residues to prevent cross-linking.

The in vitro method is also suitable for producing monoclonal antibodies. Generation of fragments thereof, particularly Fab fragments, by digestion of the antibody can be accomplished using conventional techniques known in the art.

The anti-Exo protein antibody of the present invention may further consist of a humanized antibody or a human antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof containing minimal sequence derived from non-human immunoglobulin (Fv, Fab, Fab ', F ( ab ') ₂ or an antigen-binding subsequence of an antibody). Humanized antibodies are those in which the residues from the recipient's complementarity determining regions (CDRs) are derived from CDRs of a non-human animal species such as mouse, rat or rabbit that have the desired specificity, affinity and binding ability. residue
Human immunoglobulin (recipient antibody) substituted with (donor antibody). In some cases, human immunoglobulin Fv framework residues are replaced with corresponding non-human residues. Humanized antibodies may also consist of residues that are found neither in the recipient antibody nor in the imported CDRs or framework sequences. Generally, a humanized antibody is such that all or substantially all of the CDR regions correspond to CDR regions of non-human immunoglobulin and all or substantially all of the FR regions are FR regions of human immunoglobulin consensus sequences. It will consist of at least one, and typically two, of substantially the entire variable domain. Humanized antibodies also optimally consist of at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321: 522-525 (1988). Riechmann e
t al., Nature, 332: 323-329 (1986); and Presta, Curr, Op. Struct. Biol. 2
: 593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced into them from a source other than human. These non-human amino acid residues are often referred to as "import" residues, which are typically incorporated from the "import" variable domain. Humanization is basically Winter
And co-workers (Jones et al., Nature, 321: 522-525 (1986); Riechmann et
al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-15.
36 (1988)) and replace the corresponding sequences of the human antibody with rodent CDRs or CDR sequences. Accordingly, such "humanized" antibodies are chimeric antibodies (US Pat. No. 4,816,567), which are much shorter than the original human variable domain by corresponding sequences from non-human animal species. Has been replaced. In practice, humanized antibodies are typically humanized antibodies in which some CDR residues and optionally some FR residues have been replaced with residues at similar sites in rodent antibodies. .

Human antibodies were also used in phage display libraries [Hoogenboom and Winter.
, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1
991)] and can be produced using various techniques known in the art. The techniques of Cole et al. And Boerner et al. Can also be used to prepare human monoclonal antibodies [Cole et al.
., Monoclonal Antibodies and Cancer Therapy, Alan R Liss, p. 77 (1985) an
d Boerner et al., J. Immunol., 147 (1): 86-95 (1991)]. Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals in which the endogenous immunoglobulin genes are partially or completely inactivated, such as mice. Upon antigen attack, production of human antibodies is observed, a situation very similar to that observed in humans in all respects including gene rearrangement, assembly and antibody repertoire. This approach is described, for example, in US Pat. No. 5,545,
807; 5,545,806; 5,569,825; 5,625,126; 5,633,
425; 5,661,018 and in the following scientific literature: Marks et.
al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368, 856.
-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Natu
re Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14,
826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13, 65-93 (1995).
.

Bispecific antibodies are preferably human or humanized monoclonal antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the Exo protein and the other one may be for any antigen, but is preferably for a cell surface protein, receptor or receptor subunit.

Methods for making bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two pairs of immunoglobulin heavy / light chains, where the two heavy chains have different specificities. [Milstein and Cuello
, Nature, 305: 537-539 (1983)]. Because of the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) potentially produce 10 different antibody molecules, of which only one has the correct bispecific structure. is doing. Purification of the correct molecule is usually accomplished by affinity chromatography steps. A similar procedure was published on May 13, 1993 in WO 93/08829 and Traunecker et al., EMBO J., 10: 3655-3659.
(1991).

The variable domains of antibodies with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion is preferably
The constant domain of an immunoglobulin heavy chain consisting of at least part of the hinge, CH2 and CH3 regions. It is also preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions attempted.
The DNA encoding the immunoglobulin heavy chain and, optionally, the immunoglobulin light chain are inserted into separate expression vectors and cotransfected into a suitable host organism. For details of the bispecific antibody production method, see Suresh et al., M.
See ethods in Enzymology, 121: 210 (1986).

Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies may, for example, target undesired cells to cells of the immune system [US Pat. No. 4,676,980] and for the treatment of HIV infection [WO 91/00360; WO 92/2003].
73; EP 03089]. This antibody was tested in vitro using known methods of synthetic protein chemistry, including those using cross-linking reagents.
It is being considered to be prepared at o. For example, using a disulfide exchange reaction,
Alternatively, the immunotoxin could be constructed by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate, methyl-4-mercaptobutyrimidate, and, for example, US Pat.
The reagents disclosed in No. 76,980 are mentioned.

The anti-Exo protein antibody of the present invention has various uses. For example, an anti-Exo protein antibody can be used in a diagnostic assay for Exo protein, and its expression can be detected, for example, in specific cells, tissues or serum.
A variety of diagnostic assay techniques known in the art can be used, such as competitive binding assays performed in heterogeneous or homogeneous phase, direct or indirect sandwich assays, and immunoprecipitation assays [Zola, Monoclonal Antibodies; a Manual. o
f Techniques, CRC Press, Inc. (1987) pp.147-158]. The antibody used in the diagnostic assay can also be labeled with a detectable moiety. The detectable component must be capable of producing a detectable signal, either directly or indirectly. For example, the detectable moiety may be a radioisotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I, a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine or luciferin, or alkaline phosphatase, beta-galactosidase, Alternatively, an enzyme such as horseradish peroxidase may be used. Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistry,
13: 1014 (1974); Pain et al., J. Immunol. Meth. 40: 219 (1981); and Nygre.
Any method known in the art for conjugating an antibody to a detectable moiety can be used, including the method described in n, J. Histochem. and Cytochem., 30: 407 (1982).

Anti-Exo protein antibodies are also useful for affinity purification of Exo protein from recombinant cell cultures or natural sources. In this operation,
Antibodies to Exo protein are immobilized on a suitable support such as Sephadex resin or filter paper using methods well known in the art. The immobilized antibody is then contacted with the sample containing the Exo protein to be purified, and then the support is loaded with the Exo in the sample.
Wash with a suitable solvent that removes virtually all material except proteins. Finally,
The support is washed with another solvent that releases the Exo protein from the antibody.

Anti-Exo protein antibodies can also be used therapeutically. In one embodiment, a gene encoding an antibody that binds to and regulates the action of Exo protein in cells is provided.

In one embodiment, a therapeutically effective dose of Exo protein, agonist or antagonist is administered to the patient. Here, "therapeutically effective dose" means
It refers to the dose that exerts the intended effect when administered. The exact dose depends on the purpose of treatment and can be ascertained by one skilled in the art using known techniques. Exo degradation, systemic distribution vs. local delivery, and rate of de novo protease synthesis, as well as age, weight, general health, sex, diet, time of administration, drug interaction and severity of symptoms, as known in the art. It will be necessary to make adjustments to, but one of ordinary skill in the art will be able to ascertain by routine experimentation.

“Patient” for the purposes of the present invention includes humans and other animals, especially mammals, and organisms. Thus, these methods are applicable to both human therapeutic and veterinary applications. In the preferred embodiment, the patient is a mammal, and in the most preferred embodiment the patient is human.

Administration of the Exo protein, or agonist, antagonist of the present invention includes oral, subcutaneous, intravenous, nasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular administration. It can be carried out by various methods, but is not limited to these methods. In some cases, Exo can also be administered directly as a solution or spray, eg, in the treatment of wounds and inflammation.

The pharmaceutical composition of the invention comprises the Exo protein in a dosage form suitable for administration to a patient,
It consists of agonists or antagonists. In a preferred embodiment, the pharmaceutical composition is in a water-soluble form, for example present as a pharmaceutically acceptable salt, which is meant to include both acid and base addition salts. “Pharmaceutically acceptable acid addition salts” refers to salts that retain the biological potency of the free base and are not biologically or otherwise harmful, but as examples of the acids that form them. Is an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. and acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid,
Examples thereof include organic acids such as fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid and salicylic acid. “Pharmaceutically acceptable base addition salt” includes sodium,
Potassium, lithium, ammonium, calcium, magnesium, iron, zinc,
Those derived from inorganic bases such as copper, manganese, aluminum salts and the like can be mentioned. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Examples of pharmaceutically acceptable organic non-toxic bases include:
Salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine and tripropylamine. To be

Pharmaceutical compositions may also contain one or more of the following: carrier proteins such as serum albumin; buffers; microcrystalline cellulose, lactose, corn and other starches. Excipients; binders; sweeteners and other fragrances: colorants; and polyethylene glycol. Additives are well known in the art and are used in various formulations.

All references are incorporated herein by reference in their entirety. The following examples are merely illustrative.

EXAMPLE Yeast two-hybrid system cDNA cloning technology is powerful in vi
vo protein-protein interaction assay, Fields S, Song O (1989
) Nature 340: 245-247 (Chevray PM, Nathans D (1992) Proc Natl Acad Sci USA
89: 5789-5793; Chien CT, Bartel PL, Sternglanz R, Fields S (1991) Proc N
atl Acad Sci USA 88: 9578-9582; Durfea T, Bacherer K, Chen PL, Yeh SH, Y
ang Y, Kilburn AE, Lee WH, Elledge S (1993) Genes Dev 7: 555-569; Fields
S, Song O (1989) Nature 340: 245-247; Mendelsohn AR, Brent R (1994) Bio
technology 5: 482-486; Zervos A, Gyuris J, Brent R (1993) Cell 72: 223-2
First introduced by 32). This method is based on the co-expression of two proteins X and Y fused to the DNA binding domain of GAL4 (GAL4B) and the transcriptional activation domain of GAL4 (GAL4A), respectively (FIG. 1A). If protein X interacts with protein Y, the transcriptional activation domain of GAL4
Of the reporter gene HIS3 or lacZ to activate the transcription of the reporter gene HIS3 or lacZ. The two-hybrid system can be used to clone a cDNA encoding a novel protein that interacts with a known protein (bait) in yeast. This method is also 2
It can also be used to study protein-protein interactions between a variety of known proteins. The yeast two-hybrid system has several advantages over other conventional methods for studying protein-protein interactions.
That is, protein-protein interactions are studied in eukaryotes (yeast), cD
NA clones are obtained immediately after screening, and so on.

Screening is extremely quick and convenient. No protein purification is required. Both proliferation selection and lacZ color selection are sensitive. Does not require radioisotopes. However, this technique is difficult for new users due to certain technical difficulties: the false positive rate varies significantly depending on the decoy protein used in the screen. It is difficult to control the transformation efficiency of yeast.

High quality cDNA libraries can be very difficult to construct. The major difference between the yeast one-hybrid system and the yeast two-hybrid system is that the one-hybrid system is used to clone a cDNA that encodes a protein bound to DNA but not to a protein bound to protein. That is (Fig. 1B). The DNA sequence of interest is inserted upstream of the minimal promoter that controls the expression of the HIS or lacZ gene. The cDNA fragment is fused to the C-terminus of the transcriptional activation domain of GAL4 (GAL4A) to construct a cDNA library. If the protein encoded by this cDNA can bind to a specific DNA sequence of interest, HIS / lacZ transcription is activated. The yeast one-hybrid system is widely used for cloning new transcription factors (Lehming N, Thanos D, Brickman JM, Ma J, Maniatis T, Ptashne M (
1994) Nature 371: 175-179; Li JJ, Herskowitz I (1993) Science 262: 1870-
1873; Luo Y, Stile J, Zhu L (1996) Bio Techniques 20: 564-568; Shang J,
Luo Y, Clayton D (1997) Developmental Dynamics 209: 242-253; Strubin M,
Newell JW, Matthias P (1995) Cell 80: 497-506; Wilson TE, Fahmer TJ, Joh
ston M, Milbrandt J (1991) Science 252: 1296-1300; Wang MM, Reed RR (199
3) Cell 74: 205-214). The experimental protocol between these two methods is very similar except for one notable difference. The yeast reporter strain of the one-hybrid system must be constructed by individual investigators. Reporter gene (HI
S / lacZ) expression of GAL4 in the yeast two-hybrid system
It must be under the control of the particular DNA sequence of interest, not the NA binding site. The cDNA library used for the two-hybrid screen can also be used for the one-hybrid screen.

A flow chart of the yeast two-hybrid cDNA screening experiment is outlined in FIG. A flow chart of the yeast one-hybrid cDNA screening experiment is outlined in Figure 3.

Experimental Materials Medium and Yeast Strains YPD, YPD Agar, DOB, DOBA, CSM-TRP, CSM-LEU
, CSM-HIS, CSM-URA, CSM-LYS, CSM-LEU-TRP
, CSM-LEU-HIS and CSM-LEU-TRP-HIS are all available from Bio101. 3AT (3-amino-1,2,4-triazole) is a product of Sigma (catalog number: A-8056, St., Missouri, USA).
． From Louis). Yeast two-hybrid system reporter strain Y190 (MATa, ura3-
52, his3-200, lys2-801, ade2-101, trp1-9.
01, leu2-3, 112, gal4Δ, gal80Δ, cyhr2, LYS
_{2 :: GAL1 UAS -HIS3 TATA -HIS3,} URA3 :: GAL1 U AS -GAL1 TATA -lacZ) and yeast one - hybrid system reporter strain YM4271 (MATa, ura3-52, his3-200, lys
2-801, ade2-101, trp1-903, leu2-3, 112, t
yr1-501) is Clontech Laboratories (catalog number K1603-1, Clontech, Palo Alt, CA, USA).
o).

Plasmid and cDNA Libraries The pAS2 and pACT2 series were originally developed by Elledge's laboratory (Durf.
ee et al., 1993), and Clontech laboratori.
es company (catalog numbers K1604-A, K1604-B). pGB
Other GAL4-based two-hybrid systems, such as T9 and pGAD424, were first published in the Fields lab and were published by Stratagen.
Company e (Catalog No. 235700, 235722) and Clontech la
available from the companies Borroweries (catalog numbers K1605-A, K1605-B). Two-hybrid vectors based on LexA are derived from Origene Te
It can be obtained from the company Chologies (catalog numbers DPL-100, DPL-102). C for two-hybrid and one-hybrid screening
DNA libraries are Origene, Stratagene, Clont
Available from ech and Invitrogen. Rigel also produces its own unique two-hybrid cDNA library from various tissues. All of these two-hybrid systems share the basic structure as shown in Figure 4A.

The cDNA library must be amplified before screening. Grow 10 million individual cDNA clones using at least 200 15 cm-plates. High quality plasmids can be obtained with the Qiagene DNA preparation kit.

Single-stranded carrier DNA for transformation of yeast is available from Origene or C
available from longtech. The carrier DNA also includes Ito et al. (Ito H, Fukad
a Y, Murata K, Kimura A (1993) Journal of Bacteriology 153: 163-168).

Dedicated buffer Z buffer pH 7.0 (per liter)

[Table 6]

Z buffer + X-Gal

[Table 7]

PEG / LiAc (10 ml)

[Table 8]

Equipment and Others A 30 ° C. incubator and liquid nitrogen container is required. Nylon membranes for lacZ chromogenic assay and Whatman filter papers are available from Fisher Scientific. X-Gal is a product of Promega (catalog number V3941).
, Madison, Wisconsin, USA or Denville Scie
available from Nific, Inc. (Metuchen, NJ, USA). All plastic containers are Fisher Scientific or VWR
Available from the company.

Method: Yeast Two-Hybrid System Screening Yeast reporter strains are grown from cryopreservation on YPD plates. No antibiotics are added to the yeast medium, requiring extremely rigorous aseptic manipulation during incubation. In addition, yeast strains SD-W, SD-L, SD-H, S
It is recommended to streak on DU and SD-K plates to test other yeast markers before screening the cDNA library. Reporter strains such as the Y190 strain should grow on SD-K, SD-U and SD-H plates but not on SD-W and SD-L plates. SD-H
Proliferation on plates is due to leaky expression of the HIS reporter gene. Many reporter strains are available from various sources. In general, Y190 showed higher sensitivity than other yeast strains such as HF7c. A yeast reporter strain with both the lacZ reporter gene and the HIS3 reporter gene is highly recommended. HIS selection ensures that only interaction-positive clones grow, thus facilitating colony selection later.

Determining Optimal 3AT Concentration 3AT can be used to suppress background expression of the Y190 HIS reporter gene. The concentration of 3AT varies between different reporter strains,
(HF7c) to 15 mM (Y190). To test the optimal concentration of 3AT, one yeast colony is resuspended in 10 ml TE. 100 μl of the resuspended yeast was added to SD-H + 0 mM3AT, SD-H + 5 mM3AT, S
DH + 10mM3AT, SD-H + 15mM3AT, SD-H + 25mM3A
Spread on T, and SD-H + 40 mM 3AT plates. 15mM3AT is Y
Sufficient to suppress background HIS expression of 190, but our cd
Higher concentrations of 3AT (30-40 mM) are routinely routinely used in NA library screening.

Constructing the bait plasmid The pAS2 / pACT2 series of plasmids were shown to have a higher level of sensitivity than the pGAD424 / pGBT9 series of plasmids (Estbjak J, Bren.
t R, Golemis EA (1995) Molecular and Cellular Biology 15: 5820-5829; Leg
rain P, Dokhelar MC, Transy C (1994) Nucleic Acids Research 22: 3241-324
2). The disadvantage of using pAS2 is the large size of this plasmid (8 kb)
This may make it difficult to clone large cDNA fragments into plasmids. The cDNA fragment must be fused in-frame to the C-terminus of the Gal4 binding domain (Figure 4). The junction site between Gal4 and cDNA must have the amino acid sequence GGG to avoid disruption of the domain structure. A full-length cDNA or partial fragment can be used to make a bait plasmid.

Transform bait into yeast: 1 μg of bait plasmid is transformed into Y190 using the 1 st Small Scale Yeast Transformation Protocol (see subprotocol section). Transformants are SD-W, SD
-WH, and SD-WH + 3AT (5-40 mM) plates. The LacZ chromogenic assay may be performed after colonies have grown to a diameter of 1 mm. If 3
Colonies grew on SD-WH + 40mM 3AT plates after day of incubation and / or the lacZ chromogenic assay of these colonies was X-.
If only a 30 minute incubation with Gal gave a positive result, the bait gene should be determined to be unsuitable for two-hybrid screening without further modification. The decoy gene itself is the reporter gene HIS / lac
It may be possible to activate Z. Although it is possible to perform co-transformation of the bait plasmid and cDNA in one step,
The efficiency of co-transformation is at least 10-fold lower than that of plasmid alone. The mating approach can also be used to introduce cDNA into yeast cells containing the bait vector. A protocol announced by Finley and Brent (Finley R, Bren
t R (1994) Proc Natl Acad Sci USA 91: 12980-12984).

Transforming the cDNA library: For the second transformation with the second round cDNA plasmid, grow Y190 containing the bait plasmid (see subprotocol section). The incubation time after transformation is very different from 4 to 11 days.

Identifying Positive Clones Identification of positive clones requires experience. It should also be pointed out that background colonies grow large in areas with low cell numbers on the plate, occasionally reaching the size of positive colonies in dense areas on the same plate.
The size of positive colonies must be at least 4 times larger than nearby background colonies. Also, positive colonies will turn red more quickly.

Positive colonies to be subjected to the lacZ chromogenic assay are restreaked on another SD-LWH + 3AT plate, and single colonies are isolated by the chromogenic assay for plasmid recovery. See subprotocol section for protocol of lacZ chromogenic assay. If the colonies do not turn blue after 4 hours of incubation, it is unlikely that strong protein-protein interactions are occurring. Picking up positive colonies after 12 hours of incubation is not recommended unless one is aware that the protein-protein interaction under investigation is very weak.

[0399] To recover the plasmid from the yeast to recover plasmid There are several ways from lysis by lyticase up to the glass bead method. The glass bead method is described in the subprotocol section. The electroporation method is by far the most efficient method for transforming plasmids from yeast minipreps into E. coli. Decoy and cdn
The A plasmid may have different antibiotic selectable markers to facilitate separation in E. coli. For example, the Rigel decoy plasmid has the Kan ^r gene and the cDNA plasmid has the Amp ^r gene.

Validate Positive Clones cDNA clones recovered from positive HIS / lacZ colonies must be re-transduced into yeast with other non-specific bait controls to ensure non-specific binding. In vitro binding and functional assays should also be performed to exclude false positive clones.

Engineering: Yeast One-Hybrid System Screening Selecting the well-characterized DNA sequences that construct the HIS and lacZ reporter plasmids is the most important step of the one-hybrid screen. Many DNA sequences significantly increase the basal expression level of the yeast reporter gene in the absence of the cDNA library. Multiple copies (~ 3) of the desired DNA sequence are detected in the HIS reporter plasmid pH.
ISi1 and pLacZi (Clontech Catalog No. K1603-1,
It must be inserted at both multiple cloning sites (Fig. 4B).

Grow Yeast Reporter Strains from Cryopreservation on YPD Plates Also use yeast reporter strains SD-W, SD-L, SD-H, SD-U and S.
It is recommended to streak on DK plates to test other yeast markers before screening the cDNA library. Reporter strains such as YM4271 should be able to grow on SD-K, SD-U, SD-H, SD-W, and SD-L plates.

Incorporation of HIS Reporter into Yeast To facilitate integration of the pHISi reporter into the yeast chromosome, pH
ISi must be linearized at the Xho I site. Since pHIS-1 does not have a yeast origin of replication and cannot survive in yeast without being integrated, it is not necessary to gel-purify the digested plasmid. 1 μg of digested plasmid is transformed into YM4271 using the small scale yeast transformation protocol (see subprotocol section). Use more plasmids if integration efficiency is low. Transformants were placed on SD-H plates and different concentrations of 3AT (5-5).
40 mM) on SD-H plates and incubated at 30 ° C. for at least 4 days.

Determining the Optimal 3AT Concentration If more than 40 mM is required to suppress transformant growth, pHISi
The DNA sequence inserted in is not suitable for one-hybrid screening. Note: The incorporation efficiency of pHISi is very low. 2 per SD-H plate
0-100 colonies are expected.

Colonies are picked from SD-H plates from step 3 of integrating LacZ reporter plasmid into yeast and frozen as sole HIS reporter strain YM4271 / H. pLacZi is linearized at the Nco 1 site. 1 μg of the linearized plasmid is transformed into yeast YM4271 / H and the transformants are plated on SD-U plates. Incorporation at this stage is very efficient.
It can be expected that hundreds to thousands of colonies grow on each SD-U plate. Pick up colonies and freeze as YM4271 / HB.

Large Scale Yeast Transformation Protocol with 100-200 μg of cDNA Library Screening for DNA Binding Proteins Using cDNA Library
(See subprotocol section) is used to transform YM4271 / HB. Transformants must be plated on SD-LH + 3AT (concentration determined in step 4).

Same as step 6 of the two-hybrid screening procedure to identify positive clones .

Same as step 7 of procedure for two-hybrid screening with lacZ chromogenic assay .

Same as step 8 of procedure for two-hybrid screening to recover cDNA plasmids .

Validating Positive Clones It is necessary to perform DNA gel retardation assays and other functional assays to validate the results of the 1-hybrid screens.

Sub-Protocol Small Scale Yeast Transformation (10 ⁶ Transformants / μg DNA) One yeast colony in 100 ml YPD (no plasmid) or corresponding selection medium (SD-W in the case of Y190 containing pAS2). Inoculate into a shaker at 30 ℃ 1
Incubate at 240 rpm at night. Check OD _{600 the} next day. If the OD ₆₀₀ is between 0.6 and 1.0, yeast can be used for competent cell preparation. O otherwise
Dilute to a D ₆₀₀ = 0.4 and grow for a further 3-4 hours. Cells are centrifuged in two 50 ml plastic tubes at 3000 rpm for 5 minutes. Remove the medium. Add 30 ml TE pH 7.5 and resuspend the cell pellet by vortexing rapidly. Combine the cell pellets. The cells are centrifuged again at 3000 rpm for 5 minutes. Remove TE. Estimate the amount of cell pellet and add TE to a total volume of 0.9 ml.
Completely resuspend cells by pipetting up and down. Add 100 μl of 1 M LiAc and mix well by pipetting. Competent cells are completed. Note: Competent cells do not show a significant reduction in transformation efficiency at room temperature for several hours and only a slight reduction in transformation efficiency at 4 ° C overnight. Mix 1 μg of plasmid with 10 μg / ml carrier DNA in a clean Eppendorf tube. 100 μl of competent cells from step 8 are added to this Eppendorf tube and mixed well with DNA. Add 600 μl PEG / LiAc and mix well. Note: PEG / LiAc is freshly prepared. If the transformation efficiency is not particularly important, a pre-mixed PEG / LiAc solution which has been up for less than 2 weeks can also be used. Let stand or shake and incubate at 30 ° C. for 30 minutes. Add 70 μl DMSO and mix well. Incubate in 42 ° C water bath for 15 minutes. Place on ice for 2 minutes. Centrifuge the cells in an Eppendorf centrifuge tube at 8000 rpm for 1 minute. Remove the supernatant. Resuspend the cell pellet by adding 150 μl TE. A selective medium (for example, SD-W in the case of Y190 transformed with pAS2) is plated. Incubate for 2-3 days in a 30 ° C. incubator.

Large-scale cDNA library transformation (1-10 × 10 ⁶ transformants / 100 μ
gcDNA) Yeast colony (200 ml) of YPD (single-hybrid screen)
Alternatively, the corresponding selective medium (SD-W in the two-hybrid screen) is inoculated and incubated overnight at 240 rpm in a shaker at 30 ° C. 1. Check OD _{600 the} next day. If the OD ₆₀₀ is between 0.8 and 1.0, yeast can be used for competent cell preparation. Otherwise, dilute to an OD ₆₀₀ = 0.6 and grow for an additional 3-4 hours. 2. Centrifuge cells in a 250 ml bottle at 3000 rpm for 5 minutes at room temperature. 3. Remove the medium. 4. Add 50 ml TE pH 7.5 and resuspend cell pellet by vortexing rapidly. 5. Centrifuge the cells again at 3000 rpm for 5 minutes. 6. Remove TE. 7. Repeat steps 4 to 7 again. Estimate the amount of cell pellet and add TE to a total volume of 1.8 ml. Completely resuspend cells by vortexing. Add 200 μl of 1 M LiAc and mix well by vortexing. 200 μl of 100-200 μg plasmid in a clean Eppendorf tube
Of 10 mg / ml carrier DNA. 1) DNA into competent cells while vortexing at 5000 rpm
Add drop by drop. Stir at high speed for 30 seconds to ensure thorough mixing. Add 12 ml PEG / LiAc and mix well. Note: PEG / LiAc is freshly prepared. If the transformation efficiency is not particularly important, a pre-mixed PEG / LiAc solution which has been up for less than 2 weeks can also be used. Incubate at 30 ° C. for 30 minutes with shaking. Either a rotary shaker or a rotary stirrer may be used. Add 140 μl DMSO and mix well. Incubate in 42 ° C water bath for 15 minutes. Invert several times during the incubation. Place on ice for 5 minutes to allow to cool. The cells are spun in a tabletop centrifuge at 3000 rpm for 1 minute. Remove the supernatant. 400 μl is spread on each 15 cm selective medium plate (50 plates in total). SD-LWH + 40 mM3 for two-hybrid screening of Y190 strain
Use AT plates; SD-LH + 3 for one-hybrid screening
Use AT plate. To control the transformation efficiency, 1 μl is spread on a 10 cm SD-LW plate. Incubate at 30 ° C for up to 8 days until large colonies appear.

LacZ chromogenic assay New yeast colonies are grown to a diameter of 1 mm. Fill a container (eg an ice bucket) with liquid nitrogen. Transfer the colonies from the plate using a nylon membrane. No special replica plating equipment is required. All you have to do is press the nylon membrane against the plate. Nylon membrane (catalog number N04HY08250, N04HY13250, F
Immerse Scientific Scientific, Pennsylvania, USA) in colony-side down in liquid nitrogen. Wait 20 minutes, remove the nylon membrane and dry on a paper towel for 5 minutes. In a 10 ml tube, 40 μl X-Gal is added to each 1 ml Z buffer. Add 1.5 ml Z buffer / X-Gal solution to a clean 10 cm Petri dish. To a 15 cm diameter Petri dish, add 4 ml of Z buffer / X-Gal solution. Place Whatman circular filter paper in a Petri dish and soak evenly so that all air bubbles are pushed out. Transfer the dried nylon membrane using tweezers with the colony side facing up,
Soaked Whatman circular filter paper (catalog number 09-805C, Fisher
Scientific (Pennsylvania, USA). Make sure there are no bubbles between the membrane and the round filter paper. Cover the Petri dish and incubate at 37 ° C until a blue color is visible.

Mini Isolation of Yeast Plasmids Yeast colonies are inoculated into 3 ml of selective medium (eg SD-L for cDNA library pACT). Incubate at 30 ° C. shaker or incubator overnight or until confluent. The yeast is centrifuged at 3000 rpm at room temperature in a tabletop centrifuge. The medium is removed and the pellet is resuspended in 200 μl lysis buffer. Transfer to an Eppendorf tube. Add 200 μl volume of glass beads. Note: The lid of the Eppendorf tube can be used as a spoon to scoop 200 μl glass beads. 200 μl phenol / chloroform / isoamyl alcohol (25:24
1) is added. Stir for 3 minutes at maximum vortex speed. Centrifuge at 14000 rpm for 10 minutes in a microcentrifuge. Transfer the aqueous layer to another Eppendorf tube and add 20 μl of 3M NaAc and 500 μl ethanol. Immediate precipitation should be observed. Place the Eppendorf tube in the dry ice bath for 15 minutes or until frozen. Centrifuge at 14000 rpm for 10 minutes in a microcentrifuge. Remove the supernatant and dry the pellet. The pellet is washed with 100 μl 80% ethanol and the pellet is dried in air. The pellet is resuspended in 30 μl H ₂ O and 1 ml of it is used to transform E. coli by electroporation.

Results of Two-Hybrid Screening The bait peptide was cloned into pAS2-1 and the proteins that bind to it were screened. The results are shown below.

[Table 9] PCNA is a published decoy peptide binding protein. Results of yeast one-hybrid screening have been published previously (Luo Y, Stile J, Zhu L (19
96) Bio Techniques 20: 564-568).

[Sequence list]

[Brief description of drawings]

FIG. 1A shows the basic mechanism of the yeast two-hybrid and yeast one-hybrid systems.

FIG. 1B shows the basic mechanism of the yeast two-hybrid and yeast one-hybrid systems.

FIG. 2 shows a schematic of the yeast two-hybrid screen.

FIG. 3 shows an outline of yeast one-hybrid screening.

FIG. 4A shows a two-hybrid vector.

FIG. 4B shows a two-hybrid vector.

FIG. 4C shows a 1-hybrid reporter vector.

FIG. 4D shows a 1-hybrid reporter vector.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 15/08 A61P 17/02 4C084 15/14 25/14 4C086 17/02 25/16 4C087 25/14 25 / 18 4H045 25/16 25/28 25/18 29/00 25/28 37/08 29/00 43/00 111 37/08 C07K 14/47 43/00 111 16/18 C07K 14/47 C12N 1/15 16/18 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 A C12P 21/02 G01N 33/15 Z C12Q 1/02 33 / 50 Z 1/68 33/53 D G01N 33/15 A61K 31/7125 33/50 35/76 33/53 48/00 // A61K 31/7125 C12P 21/08 35/76 C12N 15/00 ZNAA 48/00 5/00 A C12P 21/08 A61K 37/02 (31) Priority claim number 60 / 117,308 (32) Priority date 1999 January 26 (1999.26.1999) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 117,312 (32) Priority date January 26, 1999 (1999.1) 26. (33) Priority claiming country United States (US) (31) Priority claim number 60 / 117,309 (32) Priority date January 26, 1999 (Jan. 26, 1999) (33) Priority Claiming country United States (US) (31) Priority claim number 60 / 118,178 (32) Priority date February 1, 1999 (1999.2.1) (33) Priority claiming country United States (US) (31) ) Priority claim number 60 / 118,179 (32) Priority date February 1, 1999 (Feb. 1999) (33) Country of priority claim United States (US) (31) Priority claim number 60/118 , 177 (32) Priority date February 1, 1999 (February 1, 1999) (33) Priority claiming country United States (US) (31) Priority claim number 60/119, 286 (32) Priority date Heisei February 9, 2011 (February 9, 1999) (33) Priority claiming country United States (US) (31) Priority claiming number 60 / 119,998 (32) Priority date February 11, 1999 (Feb. 11, 1999) (33) Priority claiming country United States (US) (31) Priority claim number 60 / 119,759 (32) Priority date February 11, 1999 (February 11, 1999) (33) Priority claiming country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM ), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, S, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZWF F-term (reference) 2G045 AA40 DA36 FB03 4B024 AA01 AA11 BA21 BA41 CA04 DA12 EA04 GA11 HA01 4B063 QA01 QQ08 QQ43 QR32 QR01 QR24 QS34 QS39 4B064 CA72B0A4 CC02 CA27 DA24 CA02 DA27 CA24 AA99Y AB01 AC14 BA02 BA03 CA24 CA25 CA44 4C084 AA02 AA03 AA07 AA13 AA17 BA44 CA01 CA18 CA53 MA13 MA17 MA21 MA27 MA31 MA34 MA52 MA56 MA58 MA59 MA60 MA63 MA66 NA14 ZA022 ZA162 A04 A01 A04 A01 A04 A04 A04 A02 A04A04 ZA112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 ZB112 Z132 MA17 MA21 MA27 MA31 MA34 MA52 MA56 MA58 M A59 MA60 MA63 MA66 NA14 ZA02 ZA16 ZA18 ZA59 ZA81 ZA89 ZB11 ZB13 ZC35 ZC41 4C087 AA01 AA02 BC83 CA09 CA12 CA20 MA13 MA17 MA21 MA27 MA31 MA34 MA52 MA56 MA58 MA59 MA60 MA41 MA41 MA45 A11 ZA15 ZA11 ZA11 ZA11 ZA11 ZA15 ZA11 ZA11 ZA11 ZA11 ZA11 BA10 CA40 DA01 DA75 DA76 EA22 EA28 FA74

Claims (25)

[Claims] 1. A recombinant nucleic acid encoding an Exo protein, which comprises SE
Q ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88
, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO
: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID
NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ
ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99,
SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID N
O: 102, SEQ ID NO: 103, SEQ ID NO: 104, SE
Q ID NO: 105, SEQ ID NO: 106, SEQ ID NO:
107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ
ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 11
2, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID
NO: 115, SEQ ID NO: 116, SEQ ID NO: 117,
SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID N
O: 120, SEQ ID NO: 121, SEQ ID NO: 122, SE
Q ID NO: 123, SEQ ID NO: 124, SEQ ID NO:
125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ
ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 13
0, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID
NO: 133, SEQ ID NO: 134, SEQ ID NO: 135,
SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID N
O: 138, SEQ ID NO: 139, SEQ ID NO: 140, SE
Q ID NO: 141, SEQ ID NO: 142, SEQ ID NO:
A recombinant nucleic acid comprising a nucleic acid that hybridizes under high stringency conditions to each of the sequences listed in 143 and their respective complements, provided that the Exo protein binds SNAP-23. 2. SEQ ID NO: 86, SEQ ID NO: 87, S
EQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 9
0, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID N
O: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ I
D NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SE
Q ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 1
01, SEQ ID NO: 102, SEQ ID NO: 103, SEQ I
D NO: 104, SEQ ID NO: 105, SEQ ID NO: 106
, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, S
EQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO
: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ
ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 1
19, SEQ ID NO: 120, SEQ ID NO: 121, SEQ I
D NO: 122, SEQ ID NO: 123, SEQ ID NO: 124
, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID
NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, S
EQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO
: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ
ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 1
37, SEQ ID NO: 138, SEQ ID NO: 139, SEQ I
D NO: 140, SEQ ID NO: 141, SEQ ID NO: 142
, Nucleic acid sequences listed in SEQ ID NO: 143 and nucleic acids that are each at least about 90% identical to their respective complements, provided that the Exo protein binds SNAP-23. Nucleic acid. 3. An expression vector comprising the recombinant nucleic acid of claim 1 or 2, operably linked to a regulatory sequence recognized by a host cell transformed with the nucleic acid. vector. 4. A host cell comprising the recombinant nucleic acid of claim 3. 5. A method for producing an Exo protein, comprising culturing the host cell according to claim 4 under conditions suitable for the expression of the Exo protein. 6. The method of claim 5, further comprising recovering the Exo protein.
The method described in. 7. A recombinant Exo protein encoded by the nucleic acid according to claim 1 or 2. 8. SEQ ID NO: 86, SEQ ID NO: 87, S
EQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 9
0, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID N
O: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ I
D NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SE
Q ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 1
01, SEQ ID NO: 102, SEQ ID NO: 103, SEQ I
D NO: 104, SEQ ID NO: 105, SEQ ID NO: 106
, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, S
EQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO
: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ
ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 1
19, SEQ ID NO: 120, SEQ ID NO: 121, SEQ I
D NO: 122, SEQ ID NO: 123, SEQ ID NO: 124
, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID
NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, S
EQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO
: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ
ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 1
37, SEQ ID NO: 138, SEQ ID NO: 139, SEQ I
D NO: 140, SEQ ID NO: 141, SEQ ID NO: 142
, A sequence selected from the group consisting of the sequences listed in SEQ ID NO: 143, and an amino acid sequence encoded by the first 100 nucleic acid residues of their respective complements, provided that the polypeptide is SNAP. -23),
Recombinant polypeptide. 9. SEQ ID NO: 86, SEQ ID NO: 87, S
EQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 9
0, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID N
O: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ I
D NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SE
Q ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 1
01, SEQ ID NO: 102, SEQ ID NO: 103, SEQ I
D NO: 104, SEQ ID NO: 105, SEQ ID NO: 106
, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID
NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, S
EQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO
: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ
ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 1
19, SEQ ID NO: 120, SEQ ID NO: 121, SEQ I
D NO: 122, SEQ ID NO: 123, SEQ ID NO: 124
, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID
NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, S
EQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO
: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ
ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 1
37, SEQ ID NO: 138, SEQ ID NO: 139, SEQ I
D NO: 140, SEQ ID NO: 141, SEQ ID NO: 142
, An amino acid sequence encoded by a nucleic acid sequence that hybridizes under high stringency conditions to a nucleic acid selected from the group consisting of the sequences listed in SEQ ID NO: 143 and their respective complements. peptide. 10. The protein specifically binds to the Exo protein according to claim 9,
Isolated polypeptide. 11. The polypeptide according to claim 10, which is an antibody. 12. The polypeptide of claim 11, wherein the antibody is a monoclonal antibody. 13. The monoclonal antibody of claim 12, wherein the antibody reduces or eliminates the biological function of the Exo protein. 14. A method for screening a bioactive agent capable of binding to an Exo protein, the method comprising binding an Exo protein to a candidate bioactive substance, and the Exo protein of the candidate bioactive substance. A method comprising measuring binding to. 15. A method of screening for agents capable of interfering with Exo-SNAP-23 binding, comprising: a) binding Exo protein, candidate bioactive agent and SNAP-23 protein; and b). A method comprising measuring the binding of the Exo protein to the SNAP-23. 16. The method of claim 15, wherein the Exo protein and the SNAP-23 are first bound. 17. A method for screening a bioactive substance capable of modulating the activity of an Exo protein, comprising the steps of: a) adding a candidate bioactive substance to cells comprising a recombinant nucleic acid encoding the Exo protein; b). A method comprising measuring the effect of a candidate bioactive agent on the cells. 18. The method of claim 17, wherein the library of candidate bioactive agents is added to a plurality of cells containing a recombinant nucleic acid encoding an Exo protein. 19. The method according to claim 17 or 18, further comprising adding a labeling agent that labels cells that undergo exocytosis. 20. The method of claim 19, further comprising separating exocytotic cells from non-exocytotic cells. 21. The method of claim 20, wherein the separating is performed by FACS. 22. A method for treating a disease associated with exocytosis, comprising:
SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:
67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID
NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ
ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, S
EQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 7
8, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID N
O: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ I
D NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SE
Q ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89
, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO
: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID
NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ
ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100
, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID
NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, S
EQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO
: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ
ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 1
13, SEQ ID NO: 114, SEQ ID NO: 115, SEQ I
D NO: 116, SEQ ID NO: 117, SEQ ID NO: 118
, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID
NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, S
EQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO
: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ
ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 1
31, SEQ ID NO: 132, SEQ ID NO: 133, SEQ I
D NO: 134, SEQ ID NO: 135, SEQ ID NO: 136
, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID
NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, S
A protein selected from the proteins encoded by the group consisting of EQ ID NO: 142 and SEQ ID NO: 143 specifically binds to SNAP-23 expressed in tissues so that the disease is ameliorated. In particular, a method comprising administering an interfering substance. 23. A method for treating a disease associated with exocytosis, comprising:
SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO:
67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID
NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ
ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, S
EQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 7
8, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID N
O: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ I
D NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SE
Q ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89
, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO
: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID
NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ
ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100
, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID
NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, S
EQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO
: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ
ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 1
13, SEQ ID NO: 114, SEQ ID NO: 115, SEQ I
D NO: 116, SEQ ID NO: 117, SEQ ID NO: 118
, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID
NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, S
EQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO
: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ
ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 1
31, SEQ ID NO: 132, SEQ ID NO: 133, SEQ I
D NO: 134, SEQ ID NO: 135, SEQ ID NO: 136
, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID
NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, S
A method comprising administering to a patient a substance that binds to a protein encoded by a sequence selected from the group consisting of EQ ID NO: 142 and SEQ ID NO: 143, to alter exocytosis. 24. A method for reducing or inhibiting exocytosis in a cell, which comprises SEQ ID NO: 65, SEQ ID NO: 66, SEQ I.
D NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SE
Q ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72
, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO
: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID
NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ
ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83,
SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:
86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID
NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ
ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, S
EQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 9
7, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID N
O: 100, SEQ ID NO: 101, SEQ ID NO: 102, SE
Q ID NO: 103, SEQ ID NO: 104, SEQ ID NO:
105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ
ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 11
0, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID
NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,
SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID N
O: 118, SEQ ID NO: 119, SEQ ID NO: 120, SE
Q ID NO: 121, SEQ ID NO: 122, SEQ ID NO:
123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ
ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 12
8, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID
NO: 131, SEQ ID NO: 132, SEQ ID NO: 133,
SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID N
O: 136, SEQ ID NO: 137, SEQ ID NO: 138, SE
Q ID NO: 139, SEQ ID NO: 140, SEQ ID NO:
141, SEQ ID NO: 142 and SEQ ID NO: 143, in which a protein encoded by a sequence selected from the group specifically binds to SNAP23 expressed in the cell so as to inhibit exocytosis. A method comprising administering an interfering substance. 25. SEQ ID NO: 65, SEQ ID NO: 66,
SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO:
69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID
NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ
ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, S
EQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 8
0, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID N
O: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ I
D NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SE
Q ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91
, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO
: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID
NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ
ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 1
02, SEQ ID NO: 103, SEQ ID NO: 104, SEQ I
D NO: 105, SEQ ID NO: 106, SEQ ID NO: 107
, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, S
EQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO
: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ
ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 1
20, SEQ ID NO: 121, SEQ ID NO: 122, SEQ I
D NO: 123, SEQ ID NO: 124, SEQ ID NO: 125
, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID
NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, S
EQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO
: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ
ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 1
38, SEQ ID NO: 139, SEQ ID NO: 140, SEQ I
D NO: 141, SEQ ID NO: 142 and SEQ ID NO: 1
A method of neutralizing the effect of a protein encoded by a sequence selected from the group consisting of 43, which comprises contacting an agent specific for said protein with an amount of said protein sufficient to effect neutralization. A method comprising: